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Biological properties of volatile oil from Brazilian brown propolis

ABSTRACT

Propolis, is a bee product collected from exudates and flower buds of several plants, has strong aroma and several biological applications. This study aimed at evaluating the chemical composition and in vitro antioxidant, antibacterial and cytotoxic properties of volatile oil from Brazilian brown propolis. It was extracted by hydrodistillation and analyzed by gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. Volatile oil from brown propolis exhibited strong antibacterial activity against H. pylori (MIC 3.25 µg/ml), Mycobacterium tuberculosis (MIC 50 µg/ml) and M. avium (MIC 62.5 µg/ml). It was evaluated in vitro for antioxidant activity by DPPH (IC50 25.0 µg/ml) and ABTS (IC50 30.1 µg/ml) methods. Its cytotoxic property was evaluated in normal (human fibroblasts, GM07429A) and tumor (MCF-7-human breast adenocarcinoma; HeLa-human cervical adenocarcinoma and M059J-human glioblastoma) cell lines. IC50 values were 81.32 µg/ml for GM07429A and 85.00, 129.40 and 84.12 µg/ml for MCF-7, HeLa and M059J cells, respectively. Three major dereplicated components of volatile oil from brown propolis were acetophenone (15.2%), nerolidol (13.3%), and spathulenol (11.6%). Our results contribute to a better understanding of the chemical and biological properties of Brazilian brown propolis and provide evidence for its potential medicinal use.

Keywords:
Apis mellifera; Resin; Balsamic substances; Volatiles; Apitherapy

Introduction

Propolis is a resinous substance that bees produce by gathering material from different types of plants. They use it as a sealant to protect their hives from invasive organisms. Major constituents of propolis include phenolic compounds which are represented by flavonoids, phenolic acids and their esters; they attribute promising biological activities, such as antimicrobial, anti-inflammatory and antioxidant ones, to propolis. Even though the chemical composition of propolis may vary, it usually comprises 50% of resin and plant balm, 30% of wax, 10% of volatile oils, 5% of pollen and 5% of other substances, such as organic residues (Wagh, 2013Wagh, V.D., 2013. Propolis: a wonder bees product and its pharmacological potentials. Adv. Pharm. Sci., http://dx.doi.org/10.1155/2013/308249.
http://dx.doi.org/10.1155/2013/308249...
).

Wide application of propolis to modern medicine has drawn increasing attention to its chemical composition. Its composition includes chemical constituents that protect organisms against chronic diseases caused by oxidative stress, such as cancer and metabolic disorders. Thus, this natural product has promising antioxidant potential since it acts as a body defense agent against free radicals that are found in all organisms (Calegari et al., 2017Calegari, M.A., Prasniewski, A.C., da Silva, C., Sado, R.Y., Maia, F.M.C., Tonial, L.M.S., Oldoni, T.L.C., 2017. Propolis from Southwest of Parana produced by selected bees: influence of seasonality and food supplementation on antioxidant activity and phenolic profile. Ann. Braz. Acad. Sci. 89, 45-55.). In addition, previous in vitro studies have demonstrated that extracts of propolis could inhibit the growth of Mycobacterium tuberculosis as well as synergise the effect of established antitubercular drugs such as isoniazid (Ali et al., 2018Ali, M.T., Blicharska, N., Shilpi, J.A., Seidel, V., 2018. Investigation of the anti-TB potential of selected propolis constituents using a molecular docking approach. Nature 8, 12238.).

The cytotoxic activity of propolis has also drawn researchers' interest worldwide, since studies have proven that it may be applied as a nutritional supplement during cancer treatments. Several reports have shown cytotoxic effects of propolis from different origins and their fractions in several cancer cell lines (Pratsinis et al., 2010Pratsinis, H., Kletsas, D., Melliou, E., Chinou, I., 2010. Antiproliferative activity of Greek propolis. Nat. Prod. Res. 13, 286-290.; Borges et al., 2011Borges, K.S., Brassesco, M.S., Scrideli, C.A., Soares, A.E.E., Tone, L.G., 2011. Antiproliferative effects of Tubi-bee própolis in glioblastoma cell lines. Genet. Mol. Biol. 34, 310-314.).

Literature has reported advances in the use of propolis and its components, mainly regarding its promising and specific anti-Helicobacter pylori activity. Some studies show that gastric cancer is strongly related to infection caused by the Helicobacter pylori; it leads to an inflammatory process, with consequent induction of oxidative stress, and to pre-neoplastic conditions (Boyanova et al., 2005Boyanova, L., Gergova, G., Nikolov, R., Derejian, S., Lazarova, E., Katsarov, N., Mitov, I., Krastev, Z., 2005. Activity of Bulgarian propolis against 94 Helicobacter pylori strains in vitro by agar-well diffusion, agar dilution and disc diffusion methods. J. Med. Microbiol. 54, 481-483.). It should be emphasized that several biological activities described by the literature refer to extracts from propolis, rather than from its volatile oils. As a result, it should also be highlighted that this study is the first report of antioxidant, anti-Helicobacter pylori, antimycobacterial and cytotoxic properties of volatile oil extracted from a type of Brazilian brown propolis.

Considering the well-known pharmacological potential of volatile oil from Brazilian brown propolis (Fernandes et al., 2015Fernandes, F.H., Guterres, Z.R., Violante, I.M.P., Lopes, T.F.S., Garcez, W.S., Garcez, F.R., 2015. Evaluation of mutagenic and antimicrobial properties of brown propolis essential oil from the Brazilian Cerrado biome. Toxicol. Rep. 2, 1482-1488.), this study aimed to evaluate the chemical composition and in vitro antioxidant, antibacterial and cytotoxic properties of samples collected in Borda da Mata, in the south of Minas Gerais (MG) state, Brazil.

Material and methods

Fresh brown propolis (500 g) produced by Apis mellifera L. in the Atlantic Forest region in Borda da Mata, MG, was purchased from Apiário Karina (Borda da Mata, MG, Brazil) on June 15th, 2017.

Volatile oil from brown propolis (EOP) was obtained by hydrodistillation for 3 h in a Clevenger-type apparatus and stored at 4 ºC up to GC-MS, GC-FID and bioassays. Gas chromatography-flame ionization detection and gas chromatography-mass spectrometry analyses were performed by Shimadzu QP2010 Plus and GCMS2010 Plus (Shimadzu Corporation, Kyoto, Japan) systems. GC-MS and GC-FID conditions and the identification of chemical constituents of EOP were carried out in agreement with the methodology proposed by Lemes et al. (2018)Lemes, R.S., Alves, C.C.F., Estevam, E.B.B., Santiago, M.B., Martins, C.H.G., Santos, T.C.L., Crotti, A.E.M., Miranda, M.L.M., 2018. Chemical composition and antibacterial activity of essential oils from Citrus aurantifolia leaves and fruit peel against pathogenic bacteria. An. Acad. Bras. Ciênc. 90, 1285-1292..

Free radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and azino-bis (ethylbenzothiazoline-6-sulfonic acid) (ABTS+) were determined by a spectrophotometric method (Justus et al., 2018Justus, B., Almeida, V.P., Gonçalves, M.M., Assunção, D.P.S.F., Borsato, D.M., Arana, A.F.M., Maia, B.H.L.N.S., Paula, J.F.P., Budel, J.M., Farago, P.V., 2018. Chemical composition and biological activities of the essential oil and anatomical markers of Lavandula dentata L. Cultivated in Brazil. Braz. Arch. Biol. Technol. 61, e18180111.), with modifications. In the DPPH assay, different concentrations of EOP in methanol (10-100 µg/ml) were added to 2 ml 0.1 mM solution of DPPH that was previously prepared and incubated in the dark for 30 min. Absorbance was recorded at 517 nm by a UV spectrophotometer. In the ABTS assay, 1980 ml diluted ABTS+ solution was added to 20 µl EOP previously diluted in ethanol and absorbance at 734 nm was measured 6 min after initial mixing. BHT was used as positive control. Assays were carried out in triplicate. Inhibition percentage was calculated as (I%) = (A0 − A/A0) × 100, where A0 is the absorbance of the control and A is the absorbance of the samples. IC50 value was calculated as the concentration of sample required to scavenge 50% of free radicals by graphing the I% versus volatile oil concentration.

Minimum inhibitory concentration (MIC in µg/ml) of EOP was calculated by the broth microdilution method on 96-well microplates. The following ATCC standard strain was used: Helicobacter pylori (ATCC 43526). An evaluation of the activity of the volatile oil with reference drugs was made by comparing bacterial growth on each plate of H. pylori. EOP was dissolved in 5% dimethyl sulfoxide to reach final concentrations ranging between 0.195 and 400 µg/ml. The inoculum was adjusted at 625 nm in a spectrophotometer to produce cell concentration equal to 5 × 105 CFU/ml. Plates were incubated in a CO2 incubator at 37 ºC for 3 days under microaerobic conditions. Tetracycline at concentrations ranging from 0.115 to 59.0 µg/ml was employed as the standard drug, incubated under the same conditions previously mentioned. After incubation, 30 µl 0.01% aqueous resazurin solution was added to each well in order to evaluated microbial growth.

Mycobacteria Mycobacterium tuberculosis H37Rv (ATCC 27294) and M. avium (ATCC 25291) were obtained from the American Type Collection (ATCC) and maintained at −80 ºC. Antimycobacterial activity of EOP was evaluated by the MIC broth microdilution method conducted on 96-well microplates. The methodology used for evaluating antimycobacterial activity of EOP was the one described by Melo et al. (2017)Melo, D.C., Miranda, M.L.D., Júnior, W.G.F., Andrade, P.M., Alcoba, A.E.T., Silva, T.S., Cazal, C.M., Martins, C.H.G., 2017. Anticariogenic and antimycobacterial activities of the essential oil of Siparuna guianensis Aublet (Siparunaceae). Orbital: electron. J. Chem. 9, 55-60..

In this study, three different human tumor cell lines were used: breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) and glioblastoma (M059 J) (Table 3). A normal human cell line (fibroblasts, GM07492A) was included to evaluate whether EOP has selective activity. The methodology used for evaluating cytotoxic activity of EOP was the one described by Silva et al. (2019)Silva, E.A.J., Estevam, E.B.B., Silva, T.S., Nicolella, H.D., Furtado, R.A., Alves, C.C.F., Souchie, E.L., Martins, C.H.G., Tavares, D.C., Barbosa, L.C.A., Miranda, M.L.D., 2019. Antibacterial and antiproliferative activities of the fresh leaf essential oil of Psidium guajava L. (Myrtaceae). Braz. J. Biol. 79, 697-702..

Table 1
Chemical composition of volatile oil from brown propolis collected in the Atlantic Forest biome, in southeastern Brazil.
Table 2
In vitro antioxidant and antibacterial activities of volatile oil from brown propolis (IC50 and MIC).
Table 3
Cytotoxic activity (IC50 values) of volatile oil from brown propolis against different human cell lines.

Results and discussion

Brown propolis collected in southeastern Brazil, yielded high content of volatile oil (0.8%) by hydrodistillation. To identify the constituents of EOP, GC-FID and GC-MS analyses were performed and Table 1 shows the 34 constituents with relative amounts above 0.1%, which corresponds to 93.5% of identified constituents. Acetophenone (15.2%), nerolidol (13.3%), β-caryophyllene (5.9%), spathulenol (11.6%) and δ-cadinene (5.2%) were the major components of EOP. Only nerolidol and spathulenol were found in high amounts in EOP in Mato Grosso do Sul state, Brazil; acetophenone was not identified (Fernandes et al., 2015Fernandes, F.H., Guterres, Z.R., Violante, I.M.P., Lopes, T.F.S., Garcez, W.S., Garcez, F.R., 2015. Evaluation of mutagenic and antimicrobial properties of brown propolis essential oil from the Brazilian Cerrado biome. Toxicol. Rep. 2, 1482-1488.). On the other hand, only three major constituents were identified in EOP found in Piauí, in northeastern Brazil: β-caryophyllene, caryophyllene oxide and viridiflorol (Sousa et al., 2006Sousa, S.A.A., Citó, A.M.G.L., Lopes, J.A.D., 2006. Constituintes do óleo essencial da própolis produzida na cidade de Pio IX - piauí. Rev. Bras. Pl. Med. 8, 1-3.).

Concentrations of volatile compounds were found to be quite different from the ones found in the literature. It may be explained by the high variability found in plant species that grow in regions close to beehives, since bees collect plant exudates. Propolis composition depends on seasonality, illumination, altitude, collector type, food availability and the activity developed during propolis exploration. These factors influence the chemical composition of oil extracted all over the world directly; as a result, these differences are believed to contribute significantly to chemical properties and beneficial effects of all types of propolis (Toreti et al., 2013Toreti, V.C., Sato, H.H., Pastore, G.M., Park, Y.K., 2013. Recent progress of propolis for its biological and chemical compositions and its botanical origin. Evid. Complement. Alternat. Med., http://dx.doi.org/10.1155/2013/697390.
http://dx.doi.org/10.1155/2013/697390...
). Specifically, propolis explored from the hive may contain a mixture of resins from various plant sources and beeswax. If individual sources of resin are needed for chemical analysis, it may be necessary to collect the resin from plant tissue or from the hindlegs of returning resin foragers (Bankova et al., 2016Bankova, V., Bertelli, D., Borba, R., Conti, B.J., Cunha, I.B.S., Danert, C., Eberlin, M.N., Falcão, S.I., Isla, M.I., Moreno, M.I.N., Papotti, G., Popova, M., Santiago, K.B., Salas, A., Sawaya, A.C.H.F., Schwab, N.V., Sforcin, J.M., Finstrom, M.S., Spivak, M., Trusheva, B., Vilas-Boas, M., Wilson, M., Zampini, C., 2016. Standard methods for Apis mellifera propolis research. J. Apic. Res. 2, 1-49.).

Regarding antioxidant activity, results show that EOP exhibited significant DPPH free radical activity, with IC50 values of 25.0 µg/ml. When tested by the azino-bis (ethylbenzothiazoline-6-sulfonic acid) (ABTS+) method, the value of IC50 = 30.1 µg/ml was a little higher. The positive control was BHT (butylated hydroxytoluene), whose IC50 = 19.3 µg/ml (Table 2).

Antioxidant activity of EOP determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) and azino-bis (ethylbenzothiazoline-6-sulfonic acid) (ABTS+) methods were considered significant by comparison with the positive control BHT (butylated hydroxytoluene), whose IC50 was 19.3 µg/ml. BHT was chosen to be the positive control because it inhibits both free radical formation and lipid peroxidation effectively (Bajpai et al., 2017Bajpai, V.K., Baek, K.H., Kang, S.C., 2017. Antioxidant and free radical scavenging activities of taxoquinone, a diterpenoid isolated from Metasequoia glyptostroboides. S. Afr. J. Bot. 111, 93-98.). The good antioxidant activity exhibited by EOP may be explained by its major constituents, mainly the sesquiterpene spathulenol (Nascimento et al., 2018Nascimento, K.F., Moreira, F.M.F., Santos, J.A., Kassuya, C.A.L., Croda, J.H.R., Cardoso, C.A.L., Vieira, M.C., Ruiz, A.L.T.G., Foglio, M.A., Carvalho, J.E., Formagio, A.S.N., 2018. Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw. And spathulenol. J. Ethnopharmacol. 210, 351-358.).

Antibacterial activity of EOP was evaluated against H. pylori, M. tuberculosis and M. avium strains; its MIC values were measured by the broth microdilution method. EOP proved highly active against H. pylori, M. tuberculosis and M. avium with MIC = 3.25 µg/ml, 50 µg/ml and 62.5 µg/ml, respectively (Table 2). Positive controls were tetracycline (MIC = 1.0 µg/ml) for anti-Helicobacter pylori activity and isoniazid (MIC = 1.47 µg/ml) for antimycobacterial one. This result is relevant, since previous results reinforced that volatile oils whose MIC = 250 µg/ml were considered moderately active against H. pylori (Kirmizibekmez et al., 2017Kirmizibekmez, H., Karaca, N., Demirci, B., Demirci, F., 2017. Characterization of Sideritis trojana Bornm. Essential oil and its antimicrobial activity. Marm. Pharm. J. 21, 860-865.). EOP had a greater against Helicobacter pylori activity than the volatile oil from Apium nodiflorum leaves, whose MIC = 12.5 µg/ml enabled it to be defined as having appreciable value (Menghini et al., 2010Menghini, L., Leporini, L., Tirillini, B., Epifano, F., Genovese, S., 2010. Chemical composition and inhibitory activity against Helicobacter pylori of the essential oil of Apium nodiflorum (Apiaceae). J. Med. Food 13, 228-230.). EOP has also shown promising antimycobacterial activity against Mycobacterium tuberculosis (MIC = 50 µg/ml) and M. avium (MIC = 62.5 µg/ml). According to Holetz et al. (2002)Holetz, F.B., Pessini, L.G., Sanches, R.N., Cortez, D.A.G., Nakamura, V.C., Day Son, B.P., 2002. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem. I. Oswaldo. Cruz 97, 1027-1031., natural products with MIC values below 100 µg/ml, between 100 and 500 µg/ml, from 500 to 1000 µg/ml and above 1000 µg/ml exhibit good, moderate, weak and absence of antibacterial activity, respectively.

The promising antibacterial activity exhibited by EOP may be explained by synergic or even antagonistic effects among its different compounds. Most constituents of volatile oils belong to the family terpenoids; most of them have apolar character and may easily permeate the phospholipid bilayer of biological membranes. These constituents usually exhibit antimicrobial activity because they are capable of breaking through the lipid structure, cause damage to the integrity of the membrane, unbalance intracellular pH and, consequently, lead to cell death (Raut and Karuppayil, 2014Raut, J.S., Karuppayil, S.M., 2014. A status review on the medicinal properties of essential oils. Ind. Crops Prod. 62, 250-264.).

EOP cytotoxicity was assessed against the GM07492A normal cell line, whose IC50 was 81.32 µg/ml and against MCF-7, HeLa and M059 J tumor cell lines whose IC50 were 85.00, 129.40 and 84.12 µg/ml, respectively (Table 3). The positive control was doxorubicin and IC50 values are also shown in Table 3.

Cytotoxic activity of EOP was observed against normal human and tumor cell lines employed by this study, but without selectivity. Specifically, the main mechanisms that mediate cytotoxic effects of volatile oils include the induction of cell death by activation of apoptosis and/or necrosis processes, cell cycle arrest and loss of function of volatile organelles (Raut and Karuppayil, 2014Raut, J.S., Karuppayil, S.M., 2014. A status review on the medicinal properties of essential oils. Ind. Crops Prod. 62, 250-264.). It is important to highlight that the cytotoxicity assay also showed that EOP exhibits antibacterial activity against H. pylori, M. tuberculosis and M. avium at concentrations lower (MIC = 3.25 µg/ml; MIC = 50 µg/ml; MIC = 62.5 µg/ml, respectively) than those which showed cytotoxicity in normal human fibroblasts cells (IC50 = 81.32 µg/ml). Possibly, synergistic interactions among volatile oils components which are beneficial to their different biological properties (Raut and Karuppayil, 2014Raut, J.S., Karuppayil, S.M., 2014. A status review on the medicinal properties of essential oils. Ind. Crops Prod. 62, 250-264.).

Conclusions

It is clear that studies of volatile oils from propolis are far from being exhaustive. Further research is needed to reveal their chemistry and to support their medicinal properties scientifically. Differences in the chemical composition of volatile oils extracted from propolis are observed as the result of their botanic and geographic origin. Acetophenone, nerolidol, β-caryophyllene, spathulenol and δ-cadinene were the major constituents identified in EOP. Evaluation of the antibacterial activity of EOP showed that it is significant against the bacteria H. pylori, M. tuberculosis and M. avium. The preliminary result of the antioxidant potential showed by EOP could be promising for the development of news cosmetics with antioxidant potential. In assays of cytotoxic activity, EOP proved to be cytotoxic against all cell lines under study, but it showed no selectivity to tumor cell lines. The hydrodistillation process under study was capable of extracting bioactive substances from brown propolis and originate the volatile oil which is responsible for biological properties found by this study. In short, further in vivo experiments are needed to confirm the absence of potential mutagenic risks posed by EOP from the Brazilian Atlantic Forest biome and its mechanism of action.

References

  • Ali, M.T., Blicharska, N., Shilpi, J.A., Seidel, V., 2018. Investigation of the anti-TB potential of selected propolis constituents using a molecular docking approach. Nature 8, 12238.
  • Bajpai, V.K., Baek, K.H., Kang, S.C., 2017. Antioxidant and free radical scavenging activities of taxoquinone, a diterpenoid isolated from Metasequoia glyptostroboides S. Afr. J. Bot. 111, 93-98.
  • Bankova, V., Bertelli, D., Borba, R., Conti, B.J., Cunha, I.B.S., Danert, C., Eberlin, M.N., Falcão, S.I., Isla, M.I., Moreno, M.I.N., Papotti, G., Popova, M., Santiago, K.B., Salas, A., Sawaya, A.C.H.F., Schwab, N.V., Sforcin, J.M., Finstrom, M.S., Spivak, M., Trusheva, B., Vilas-Boas, M., Wilson, M., Zampini, C., 2016. Standard methods for Apis mellifera propolis research. J. Apic. Res. 2, 1-49.
  • Boyanova, L., Gergova, G., Nikolov, R., Derejian, S., Lazarova, E., Katsarov, N., Mitov, I., Krastev, Z., 2005. Activity of Bulgarian propolis against 94 Helicobacter pylori strains in vitro by agar-well diffusion, agar dilution and disc diffusion methods. J. Med. Microbiol. 54, 481-483.
  • Borges, K.S., Brassesco, M.S., Scrideli, C.A., Soares, A.E.E., Tone, L.G., 2011. Antiproliferative effects of Tubi-bee própolis in glioblastoma cell lines. Genet. Mol. Biol. 34, 310-314.
  • Calegari, M.A., Prasniewski, A.C., da Silva, C., Sado, R.Y., Maia, F.M.C., Tonial, L.M.S., Oldoni, T.L.C., 2017. Propolis from Southwest of Parana produced by selected bees: influence of seasonality and food supplementation on antioxidant activity and phenolic profile. Ann. Braz. Acad. Sci. 89, 45-55.
  • Fernandes, F.H., Guterres, Z.R., Violante, I.M.P., Lopes, T.F.S., Garcez, W.S., Garcez, F.R., 2015. Evaluation of mutagenic and antimicrobial properties of brown propolis essential oil from the Brazilian Cerrado biome. Toxicol. Rep. 2, 1482-1488.
  • Holetz, F.B., Pessini, L.G., Sanches, R.N., Cortez, D.A.G., Nakamura, V.C., Day Son, B.P., 2002. Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem. I. Oswaldo. Cruz 97, 1027-1031.
  • Justus, B., Almeida, V.P., Gonçalves, M.M., Assunção, D.P.S.F., Borsato, D.M., Arana, A.F.M., Maia, B.H.L.N.S., Paula, J.F.P., Budel, J.M., Farago, P.V., 2018. Chemical composition and biological activities of the essential oil and anatomical markers of Lavandula dentata L. Cultivated in Brazil. Braz. Arch. Biol. Technol. 61, e18180111.
  • Kirmizibekmez, H., Karaca, N., Demirci, B., Demirci, F., 2017. Characterization of Sideritis trojana Bornm. Essential oil and its antimicrobial activity. Marm. Pharm. J. 21, 860-865.
  • Lemes, R.S., Alves, C.C.F., Estevam, E.B.B., Santiago, M.B., Martins, C.H.G., Santos, T.C.L., Crotti, A.E.M., Miranda, M.L.M., 2018. Chemical composition and antibacterial activity of essential oils from Citrus aurantifolia leaves and fruit peel against pathogenic bacteria. An. Acad. Bras. Ciênc. 90, 1285-1292.
  • Melo, D.C., Miranda, M.L.D., Júnior, W.G.F., Andrade, P.M., Alcoba, A.E.T., Silva, T.S., Cazal, C.M., Martins, C.H.G., 2017. Anticariogenic and antimycobacterial activities of the essential oil of Siparuna guianensis Aublet (Siparunaceae). Orbital: electron. J. Chem. 9, 55-60.
  • Menghini, L., Leporini, L., Tirillini, B., Epifano, F., Genovese, S., 2010. Chemical composition and inhibitory activity against Helicobacter pylori of the essential oil of Apium nodiflorum (Apiaceae). J. Med. Food 13, 228-230.
  • Nascimento, K.F., Moreira, F.M.F., Santos, J.A., Kassuya, C.A.L., Croda, J.H.R., Cardoso, C.A.L., Vieira, M.C., Ruiz, A.L.T.G., Foglio, M.A., Carvalho, J.E., Formagio, A.S.N., 2018. Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw. And spathulenol. J. Ethnopharmacol. 210, 351-358.
  • Pratsinis, H., Kletsas, D., Melliou, E., Chinou, I., 2010. Antiproliferative activity of Greek propolis. Nat. Prod. Res. 13, 286-290.
  • Raut, J.S., Karuppayil, S.M., 2014. A status review on the medicinal properties of essential oils. Ind. Crops Prod. 62, 250-264.
  • Silva, E.A.J., Estevam, E.B.B., Silva, T.S., Nicolella, H.D., Furtado, R.A., Alves, C.C.F., Souchie, E.L., Martins, C.H.G., Tavares, D.C., Barbosa, L.C.A., Miranda, M.L.D., 2019. Antibacterial and antiproliferative activities of the fresh leaf essential oil of Psidium guajava L. (Myrtaceae). Braz. J. Biol. 79, 697-702.
  • Sousa, S.A.A., Citó, A.M.G.L., Lopes, J.A.D., 2006. Constituintes do óleo essencial da própolis produzida na cidade de Pio IX - piauí. Rev. Bras. Pl. Med. 8, 1-3.
  • Toreti, V.C., Sato, H.H., Pastore, G.M., Park, Y.K., 2013. Recent progress of propolis for its biological and chemical compositions and its botanical origin. Evid. Complement. Alternat. Med., http://dx.doi.org/10.1155/2013/697390
    » http://dx.doi.org/10.1155/2013/697390
  • Wagh, V.D., 2013. Propolis: a wonder bees product and its pharmacological potentials. Adv. Pharm. Sci., http://dx.doi.org/10.1155/2013/308249
    » http://dx.doi.org/10.1155/2013/308249

Publication Dates

  • Publication in this collection
    3 Feb 2020
  • Date of issue
    Nov-Dec 2019

History

  • Received
    7 May 2019
  • Accepted
    27 July 2019
  • Published
    5 Sept 2019
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