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Antibacterial and antiproliferative activities of the fresh leaf essential oil of Psidium guajava L.

1. Introduction

Essential oils are sources of natural substances with several biological activities with antioxidant, antimicrobial, anticancer, antinociceptive, antiviral, and antiphlogistic properties (Martins et al., 2015).

Tooth decay is an important public health problem that affects a large number of people in many countries in the world. More than 700 species of bacteria are identified in the oral cavity and some of them are responsible for caries and other periodontal diseases, among them we can highlight the bacteria of the genus Streptococcus (Melo et al., 2017).

Plant-derived materials such as plant extracts, essential oils, and pure compounds have antimicrobial effects against oral pathogens and these materials have attracted the interest of researchers worldwide. However, reports on the antimicrobial activity of natural products against oral pathogens are still scarce (Estevam et al., 2016).

Currently, cancer treatment is considered one of the most challenging problems in medicine and several experimental and epidemiological studies have shown that the use of some plants may promote chemopreventive and/or antineoplastic action (Oliveira et al., 2014). In this scenario, some essential oils extracted from different plants have promising antitumor potential, both in vitro and in vivo (Lesgards et al., 2014).

The species Psidium guajava L., common name guava, belongs to the family Myrtaceae, which is composed of more than 100 genera and 3800 species and is one of the most studied species of this family (Haida et al., 2015). The essential oils and leaf extracts of this plant have several biological activities, including antidermatophytic, anti-inflammatory, antibiotic, analgesic, hepato-protective and antioxidant properties (Bhushan et al., 2014; Ferdinand et al., 2014).

This study, continuing our line of research on chemical composition and the biological activities of essential oils (Lemes et al., 2017; Oliveira et al., 2017), analyzes the chemical composition and the antibacterial and antiproliferative activities of the fresh leaf essential oil of P. guajava L. collected in the southwest region of the state of Goias.

2. Material and Methods

2.1. Plant material

The experiment was conducted at the Laboratory of Natural Products Chemistry of the Instituto Federal Goiano--Campus Rio Verde, GO. In March 2014, fresh P guajava leaves were collected at 4:00 p.m from a native population in the Rio Verde region. Collection took place at coordinates 17[degrees]48'12.006"S and 50[degrees]54'19.083"W, 715m altitude. Plant material was identified, and the samples were deposited as desiccated specimens in the Herbarium of the Universidade Estadual de Montes Claros, state of Minas Gerais, Brazil under identification number 4481.

2.2. Essential oil extraction

The essential oil was extracted from fresh leaves of P. guajava (100 g) ground in a knife mill by the hydrodistillation method using a Clevenger type apparatus at 100 [degrees]C for 4 h (Xavier et al., 2016). Thereafter, the hydrolate was subjected to liquid-liquid partition in a separatory funnel and three washes with three 10 mL portions of dichloromethane. Essential oil samples were stored at -4 [degrees]C until further chemical and biological tests.

2.3. Chemical analysis of essential oil

Essential oil chemical composition was analyzed at the Laboratory of Analysis and Synthesis of Agrochemicals of the Universidade Federal de Vicosa, Minas Gerais. Quantitative analysis of the essential oil components was performed in a Shimadzu GC-17A gas chromatograph equipped with a flame ionization detector (FID) and SPB-5 fused silica capillary column (30 m x 0.25 mm; 0.25-pm film thickness). Nitrogen was used as the carrier gas (1.8 mL [min.sup.-1]), split 1/10, and injector and detector temperatures were 220 and 240 [degrees]C, respectively. The initial column temperature was 40 [degrees]C isothermal for 4 minutes, followed by heating at 3 [degrees]C [min.sup.-1] up to 240 [degrees]C; 1[micro]L of the essential oil was injected for the analysis. Essential oil components were identified using a gas chromatograph (CG-EM Shimadzu, QP-5050A) equipped with a mass-selective detector, operating by electronic ionization (70 eV), with an RTX-5 fused silica column (30 m x 0.25 mm; 0.25-pm film thickness). Chromatographic conditions were identical to the conditions applied to the CG-FID, except for the carrier gas, which in this case was helium at a 1mL [min.sup.-1] flow. The identification of constituents was based on the retention indices relative to [C.sub.9]-[C.sub.22] alkanes and by comparing the mass spectra with a computer databank (Wiley 7 and Nist 62) and with published data (Adams, 2007).

2.4. Antibacterial assays

Bacteria were acquired from the American Type Culture Collection (ATCC) and kept in the culture collection of the Laboratory of Research on Applied Microbiology (LaPeMA) at Universidade de Franca, state of Sao Paulo, Brazil, at -80[degrees]C. The following microorganisms were used: Streptococcus salivarius (ATCC 25975), Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49452), Streptococcus sanguinis (ATCC 10556) and Streptococcus sobrinus (ATCC 33478).

Minimum Inhibitory Concentrations (MIC) of essential oils were determined by the broth microdilution method in 96-well microplates, following the methodology of CLSI (2006). Samples were dissolved in 125 [micro]L tryptic soy broth (TSB) to yield compound concentrations between 50 and 400 [micro]g/mL. The inoculum was adjusted to 625 nm for every microorganism in a spectrophotometer to obtain cell concentration of 5 x 105 colony-forming units (CFU/mL) (CLSI 2006). Chlorhexidine digluconate (Sigma-Aldrich), at concentrations from 0.115 to 59.0 [micro]g/mL, was used as the positive control. The microplates were incubated at 37 [degrees]C for 24 h; then, 30 [micro]L 0.02% resazurin (Sigma-Aldrich) aqueous solution was added to every well (Sarker et al., 2007). Resazurin is an oxireduction probe that enables microbial growth to be immediately observed. Blue and red colors represent the absence and the presence of microbial growth, respectively.

2.5. Cell lines and culture conditions

In this study, we used three different tumor cell lines: human breast adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa), and human gliobastoma (M059J). A normal human cell line (lung fibroblasts, GM07492A) was included to evaluate the possible selective activity of the natural product tested. The different cell lines were maintained as monolayers in plastic culture medium (HAM-F10 + DMEM, 1:1, Sigma-Aldrich) supplemented with 10% fetal bovine serum (Nutricell), antibiotics (0.01 mg/mL streptomycin and 0.005 mg/mL penicillin; Sigma-Aldrich), and 2.38 mg/mL Hepes (Sigma-Aldrich). The cells were incubated at 36.5 [degrees]C in a humidified 5% C[O.sub.2] atmosphere.

2.6. Antiproliferative assay

The antiproliferative activity was measured using the in vitro Toxicology Colorimetric Assay Kit (XTT; Roche Diagnostics) according to the manufacturer's instructions. For the experiments, the cells ([10.sup.4] cells/well) were plated onto 96-well microplates. Each well received 100 [micro]L HAM-F10/DMEM medium containing essential oil at concentrations ranging from 3.91 to 500 [micro]g/mL. Negative (no treatment), solvent (0.02% DMSO, dimethylsulfoxide, Sigma-Aldrich) and positive (doxorubicin, DXR, Pharmacia Brasil Ltda.,) controls were included. After incubation at 36.5 [degrees]C for 24 h, the culture medium was removed. The cells were washed with 100 [micro]L of PBS (phosphate buffered saline) to remove the treatments and exposed to 100 [micro]L of culture medium HAM-F10 without phenol red. Then, 25 [micro]L of XTT was added, and the cells were incubated at 36.5 [degrees]C for 17 h. The absorbance of the samples was determined using a multi-plate reader (ELISA--Tecan--SW Magellan vs 5.03 STD 2P) at the wavelength of 450 nm and reference length of 620 nm. The antiproliferative activity was assessed using [IC.sub.50], the concentration able to inhibit 50% of cell line growth as a response parameter, which was calculated with the GraphPad Prism program by plotting cell survival against the respective concentrations of the natural product tested. One-way ANOVA was used for the comparison of means (p [less than or equal to] 0.05). The experiments were performed in triplicate. The selectivity index was calculated by dividing the [IC.sub.50] value of the essential oil obtained for GM07492A cells by the [IC.sub.50] value obtained for the cancer cell line.

3. Results and Discussion

The essential oil of P. guajava fresh leaves is composed mainly by hydrocarbon sesquiterpenes (62.0%), followed by oxygenated sesquiterpenes (14.8%), hydrocarbons monoterpenes (1.8%), and oxygenated monoterpenes (1.2%) (Table 1). Seventeen compounds were identified in the essential oil of P. guajava, which corresponded to 94.1% of the total oil analyzed and the main components are: [beta]-caryophyllene (16.1%), [alpha]-humulene (11.9%), aromadendrene oxide (14.7%), [delta]-selinene (13.6%), and selin-11-en-4[alpha]-ol (12.5%) (Table 1).

The chemical composition of the essential oil extracted from fresh leaves of P. guajava, collected in the state of Goias, showed little similarity to other oils reported in the literature for species occurring in other countries. For example, the major constituents identified in the leaf essential oil of P. guajava from Nepal were E-nerolidol (35.6%) and E-caryophyllene (15.8%) (Satyal et al., 2015), whereas in Tunisia, the major constituents were viridiflorol (36.4%) and irans-caryophyllene (5.9%) (Khadhri et al., 2014) and, in Nigeria, the predominant constituents were limonene (42.1%) and [beta]-caryophyllene (21.3%) (Ogunwande et al., 2003). The essential oil chemical composition ofP guajava occurring in Lavras, a municipality in the southern region of Minas Gerais, showed similarity with the chemical composition found in the present study, especially the compound selin-11-en-4[alpha]-ol was present in both (Lima et al., 2010). The chemical composition observed in the present study was similar to the chemical composition already described in the literature for other species belonging to the same genus and Myrtaceae family (Scur et al., 2016).

Regarding the antibacterial activity, several papers have reported the antimicrobial potential of plant essential oils against oral pathogens over the last decade (Sousa et al., 2015). Table 2 shows the antibacterial activity of the fresh leaf essential oil of P. guajava against a representative panel of oral pathogens, with MIC values ranging from 400 to 100 [micro]g/mL.

According to Holetz et al. (2002), the antimicrobial activity can be considered good when MIC values are below 100 [micro]g/mL; moderate from 500 to 100 [micro]g/mL; weak from 1000 to 500 [micro]g/mL; and inactive above 1000 [micro]g/mL.

The PG-EO showed moderate antibacterial activity against all bacteria of the genus Streptococcus assessed in this work. This activity can be explained by the chemical constituents of known antibacterial activity such as caryophyllene oxide (4.1%), [beta]-caryophyllene (16.1%), and a-humulene (11.9%) present in the oil (Moreira et al., 2014).

Several mechanisms are proposed to explain the antimicrobial activity of essential oils. It is believed that microbial growth inhibition by essential oils is due to the direct damage to cell membrane integrity caused by their lipophilic components, which affects cell pH maintenance and inorganic ion balance. According to the literature, the inhibitory effects of essential oils are consistent with the action of monoterpene and sesquiterpene constituents on the cell membrane, and the damage to membrane produces different effects on microorganisms (Oliveira et al., 2016).

The PG-EO cytotoxicity was assessed against the GM07492A normal cell line, with an [IC.sub.50] of 126.4 [+ or -] 11.8 [micro]g/mL, and against the MCF-7, HeLa, and M059J tumor lines with [IC.sub.50] of 96.9 [+ or -] 8.4; 128.7 [+ or -] 1.5; and 103.6 [+ or -] 5.1 [micro]g/mL, respectively (Table 3). The [IC.sub.50] of the lines MCF-7 and M059J were significantly lower than that of the normal line, with selectivity indices of 1.2 and 1.3, respectively. The [IC.sub.50] values of PG-EO are lower and have higher antiproliferative activity than the [IC.sub.50] values of the leaf essential oil of Rosmarinus officinalis against the cell lines MCF-7 ([IC.sub.50] = 190.1 [micro]g/mL) and LNCaP ([IC.sub.50] = 180.9 [micro]g/mL) (Hussain et al., 2010). Hussain et al., (2010) classified [IC.sub.50] values < 10 [micro]g/mL as potentially very toxic, [IC.sub.50] from 10 to 100 [micro]g/mL as potentially toxic, [IC.sub.50] from 100 to 1000 [micro]g/mL as potentially harmful, and [IC.sub.50] > 1000 [micro]g/mL as potentially non-toxic.

The evidence from this study, the high amount of terpenes, known for their anticancer activity such as [beta]-caryophyllene (16.1%), aromadendrene oxide (14.7%), S-selinene (13.6%), selin-11-en-4[alpha]-ol (12.5%), [alpha]-humulene (11.9%), [beta]-caryophyllene oxide (4.1%), and [alpha]-cadinol (1.6%) (Table 1), suggests that P. guajava essential oil is a significant potential source of pure compounds with promising anticancer activity (Salvador et al., 2011; Quassinti et al., 2013; Fidyt et al., 2016; Guerrini et al., 2016). Besides, despite the low concentration of hydrocarbon monoterpenes (1.8%) and oxygenated monoterpenes (1.2%) in the essential oil of P. guajava (Table 1), this class of compounds deserves special attention for its important antitumor activity (Sobral et al., 2014). Furthermore, it is worth mentioning that the anticariogenic and antiproliferative activities of the leaf essential oil ofP guajava occurring in the southwestern region of Goias can also be explained by the synergistic effect among all its chemical constituents (Lesgards et al., 2014; Casanova and Costa 2017).

4. Conclusion

The GC-MS and GC-FID analysis of the essential oil of P. guajava leaves revealed the presence of seventeen compounds, among which [beta]-caryophyllene, a-humulene, aromadendrene oxide, S-selinene, and selin-11-en-4[alpha]-ol were the major components. Whit respect to testing the antibacterial activity, the essential oil showed moderate antibacterial activity against all bacteria of the genus Streptococcus assessed in this study. Regarding the anticancer activity, the essencial oil of P. guajava leaves was effective against different tumor cell lines tested.

https://doi.org/10.1590/1519-6984.189089

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E. A. J. Silva (a) *, E. B. B. Estevam (a), T. S. Silva (b), H. D. Nicolella (b), R. A. Furtado (b), C. C. F. Alves (a), E. L. Souchie (a), C. H. G. Martins (b), D. C. Tavares (b), L. C. A. Barbosa (c) and M. L. D. Miranda (d)

(a) Instituto Federal de Educacao, Ciencia e Tecnologia Goiano, Rodovia Sul Goiana, s/n, Km 1, CP 66, CEP 75901-970, Rio Verde, GO, Brasil

(b) Centro de Pesquisa em Ciencias Exatas e Tecnologia, Universidade de Franca--UNIFRAN, Av. Dr. Armando Salles Oliveira, 201, Parque Universitario, CEP 14404-600, Franca, SP, Brasil

(c) Departamento de Quimica, Universidade Federal de Vicosa, Campus Universitario, Av. Peter Henry Rolfs, s/n, 36570-900, Vicosa, MG, Brasil

(d) Instituto Federal de Educacao, Ciencia e Tecnologia do Sul de Minas Gerais, Av. Maria da Conceicao Santos, 900, Parque Real, CEP 37550-000, Pouso Alegre, MG, Brasil

* e-mail: elizabethjosefi@yahoo.com.br

Received: December 13, 2017--Accepted: March 27, 2018--Distributed: November 30, 2019
Table 1. Compounds identified in the leaf essential oil of
P. guajava (Myrtaceae).

Compounds                       RI *   RA (%)

Limonene                        1024    1.8
1,8-Cineole                     1026    1.2
[alpha]-Copaene                 1374    1.3
[beta]-Caryophyllene            1419   16.1
[alpha]-Humulene                1454   11.9
4,11-Selinadiene                1475    1.3
[gamma]-Muurolene               1478    1.4
Aromadendrene oxide             1488   14.7
[delta]-Selinene                1499   13.6
[alpha]-Panasinsene             1517    1.3
trans-Nerolidol                 1566    3.3
[beta]-Caryophyllene oxide      1585    4.1
Humulene epoxide II             1612    1.4
Longipinene epoxide             1620    1.8
epi-[alpha]-Muurolol            1639    4.0
Selin-11-en-4[alpha]-ol         1645   12.5
[alpha]-Cadinol                 1660    1.6
Total identified                       94.1
Hydrocarbon monoterpenes                1.8
Oxygenated monoterpenes                 1.2
Hydrocarbon sesquiterpenes             62.0
Oxygenated sesquiterpenes              14.8

* RI: Retention index from the literature. (%) RA: Relative area
(peak area relative to the total peak in the GC-MS chromatogram),
average of three replicates.

Table 2. Antibacterial activity of essential oil of
P. guajava leaves (PG-EO) against oral bacteria.

Minimum inhibitory concentration (MIC)--[micro]g/mL

Bacteria                   CHD *

Streptococcus        400   0.922
  salivarius (a)
S. mutans (a)        200   0.922
S. mitis (a)         200   3.688
S. sanguinis (a)     400   0.922
S. sobrinus (a)      100   0.922

(a) Aerobic Gram-positive bacteria. * CHD: chlorhexidine
dihydrochloride (positive control).

Table 3. [IC.sub.50] and selectivity index (SI) of essential
oil of P. guajava leaves (PG-EO) against diferent cell lines.

              Treatment ([micro]g/mL)

                     PG-EO

Cell line

                  [IC.sub.50]        SI

GM07492A      126.4 [+ or -] 11.8    --
MCF-7          96.9 [+ or -] 8.4a    1.3
HeLa          128.7 [+ or -] 1.5     --
M059J         103.6 [+ or -] 5.1a    1.2

               Treatment ([micro]g/mL)

                      DXR

Cell line

                  [IC.sub.50]        SI

GM07492A        0.5 [+ or -] 0.2     --
MCF-7          62.1 [+ or -] 2.0     --
HeLa            5.3 [+ or -] 1.3     --
M059J          16.2 [+ or -] 2.5     --

Doxorubicin (DXR) was used as positive controls. GM07492A
(human lung fibroblasts), MCF-7 (human breast adenocarcinoma),
HeLa (human cervical adenocarcinoma) and M059J (human
glioblastoma). The selectivity index is the ratio between the
[IC.sub.50] value of the PG-EO obtained for GM07492A cells and the
value found for the tumor cell line. Values are mean [+ or -] SD,
n = 3. Significantly different from the normal cell line (GM07492A)
(p [less than or equal to] 0.05).
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Title Annotation:Original Article
Author:Silva, E.A.J.; Estevam, E.B.B.; Silva, T.S.; Nicolella, H.D.; Furtado, R.A.; Alves, C.C.F.; Souchie,
Publication:Brazilian Journal of Biology
Date:Sep 12, 2019
Words:4412
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