Studies on the essential oils composition, antibacterial and cytotoxicity of Eugenia uniflora L.

The International Journal of Aromatherapy (2005) 15, 147–152

The International Journal of

Aromatherapy intl.elsevierhealth.com/journals/ijar

Studies on the essential oils composition, antibacterial and cytotoxicity of Eugenia uniflora L. I.A. Ogunwande a,c,*, N.O. Olawore b, O. Ekundayo c, T.M. Walker d, J.M. Schmidt d, W.N. Setzer d a

Laboratory of Food Analysis, Institute of Food Biotechnology, Department of Bioscience and Biotechnology, Division of Bioresources and Bioenvironmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi 812-8581, Japan b Department of Pure and Applied Chemistry, Ladoke Akintola University of Technology, Ogbomoso 210001, Nigeria c Department of Chemistry, University of Ibadan, Ibadan, Nigeria d Department of Chemistry, University of Alabama at Huntsville, Huntsville, AL 35899, USA

KEYWORDS

Summary The compositional profile of the essential oils isolated from Eugenia uniflora L. revealed the occurrence of an unusual sesquiterpene as the major compound. The volatile oils were characterized by the abundance of curzerene (19.7%), selina-1,3,7(11)-trien-8-one (17.8%), atractylone (16.9%) and furanodiene (9.6%) in the leaves; and germacrone (27.5%), selina-1,3,7(11)-trien-8-one (19.2%) curzerene (11.3%) and oxidoselina-1,3,7(11)-trien-8-one (11.0%) in the fruits. The two oils exhibited potent cytotoxic activity and varying antibacterial effects. c 2005 Published by Elsevier Ltd.

Eugenia uniflora; Essential oils; Sesquiterpenes; Curzerene; Germacrone; Cytotoxicity




Introduction Plants have been used for medicinal purposes since time immemorial, with the volatile oil fraction being an important economic and medicinal extract. Due to their bioactive components essential or volatile oils are becoming promising as sources of natural medicinal products. The quantitative * Corresponding author. E-mail address: [email protected] (I.A. Ogunwande).




0962-4562/$ - see front matter c 2005 Published by Elsevier Ltd. doi:10.1016/j.ijat.2005.07.004

composition and the relative proportions of the oil components are widely influenced by the genotype, ontogenic development and the environmental and growing conditions (Piccaglia et al., 1991; Shu and Lawrence, 1977). This implies the possibility of different medicinal uses of a plant species grown in different regions. Many oils have been found to be antimicrobial (Gbolade and Adebajo, 1993; Adebajo et al., 1989; Iraj and Mirmostafa, 2003; Oladimeji et al., 2004), antioxidant (Takahashi et al., 2003), anti-tumor (Legault et al., 2003), useful in therapeutic treatment (Prichard,

148 2004; Baylac and Racine, 2004; Crowell, 1999) and possess some pharmacological properties (Barbara et al., 2003). The genus Eugenia is one of the largest genera of the Myrtaceae family with ca. 500 species, most of them found growing in South America. It comprises a large group of medicinal plants with therapeutic applications. Amongst them the shrubby Eugenia uniflora, commonly referred to as ‘pitanga cherry’, is widely distributed in the tropics and subtropics. Its cherry-like fruit is edible and its leaves are used in folk medicine to lower blood glucose levels (Matsumura et al., 2003), as a diuretic, anti-rheumatic, anti-febrile, anti-inflammatory and against stomach diseases (Weyerstahl et al., 1988). Studies on the chemical composition of the plant have been focused mainly on the volatile constituents from the leaves, but a number of polyphenols and tannins have also been isolated and characterized (Lee et al., 1997). Three chemotypes of the essential oils of the plant have been reported: 1. The chemotype growing in north eastern Brazil and Nigeria that presented selina-1,3,7(11)trien-8-one and oxidoselina-1,3,7(11)-trien-8one as the main constituents (de Morais et al., 1996;Weyerstahl et al., 1988). 2. The chemotype growing in Southern Brazil that contained a-, b-selinene and nerolidol as the principal compounds (Henriques et al., 1993). 3. The chemotype reported from plants cultivated in Argentina whose main components were the monoterpenes pulegone, carvone, limonene and the sesquiterpene nerolidol (Henriques et al., 1993). The only known report concerning the chemical constituents of its fruit volatile oil identified the sesquiterpenes furanoelemene, germacrene B, celemene and selina-4(14),7(11)-diene as the main constituents (Weyerstahl et al., 1988). This paper reports that the essential oils obtained from the leaves and fruits of Eugenia uniflora possess considerable antibacterial and cytotoxic activities. This is an indication of the chemotherapeutic potentials of these volatile oils.

Materials and methods Plant material Fully grown leaves and fruits of E. uniflora were collected from plants cultivated at the campus of the University of Ibadan, Nigeria, in April 2003. Plant materials were authenticated by Mr. T.K.

I.A. Ogunwande et al. Odewo of the Herbarium Headquarters, Forestry Research Institute of Nigeria (FRIN), Ibadan, where voucher specimen (FHI 106567) was deposited.

Extraction of the volatile oils Essential oils were obtained by hydrodistillation (3 h) of the fresh plant materials using a Clevenger type apparatus in accordance with the British Pharmacopoeia (1980).

Gas chromatography-mass spectrometry analyses (GC-MS) The volatile oil samples were subjected to GC-MS analyses on an Agilent system consisting of a model 6890 Gas Chromatograph, a model 5973 Mass selective detector (MSD) and an Agilent ChemStation data system. The GC column was an HP-5ms fused silica capillary with a (5% phenyl)-methylpolysiloxane stationary phase, film thickness of 0.25 lm, a length of 30 m and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 7.07 psi and flow rate of 1.0 mL/ min. Inlet temperature was 200
C and MSD detector temperature was 280
C. The GC oven temperature program was used as follows: 40
C initial temperature, held for 10 min; increased at 3
/ min to 200
C; increased 2
/min to 220
C. The sample was dissolved in CH2Cl2 and a split injection technique was used.

Identification of the constituents Identification of each individual constituent of the essential oils was achieved based on their retention indices (determined with a reference to a homologous series of normal alkanes), and by comparison of their mass spectral fragmentation patterns (NIST database/ChemStation data system) (Adams, 2001), and with references to published literature data (Pala-Paul et al., 2001; Roussis et al., 2000; Skaltsa et al., 2003).

Cell culture media Human Hep G2 hepatocellular carcinoma cells (ATCC No. HB-8065) (Knowles et al., 1980) were grown in an air environment at 37
C in Dulbecco’s Modified Eagle’s Medium (DMEM) with L-glutamine and 1000 mg glucose per litre of medium, supplemented with 100,000 units penicillin and 10.0 mg streptomycin per litre of medium, and buffered with 30 mM N-(2-hydroxyethyl) piperazine-N0 -2ethanesulfonic acid (Hepes), pH 7.35. Cells were

Studies on the essential oils composition, antibacterial and cytotoxicity of Eugenia uniflora L. plated using medium supplemented with 10% foetal bovine serum and maintained between passaging using medium supplemented with 10% horse serum and 5% foetal bovine serum. Human Hs 578T breast ductal carcinoma cells (ATCC No. HTB-129) (Hackett et al., 1977) were grown in a 3% CO2 environment at 37
C in DMEM with 4500 mg glucose per litre of medium, supplemented with 10% foetal bovine serum, 10 lg bovine insulin, 100,000 units penicillin and 10.0 mg streptomycin per litre of medium, and buffered with 44 mM NaHCO3, pH 7.35. Human PC-3 prostatic carcinoma cells (ATCC No. CRL- 1435) (Kaighn et al., 1979) were grown in a 3% CO2 environment at 37
C in RPMI-1640 medium with L-glutamine, supplemented with 10% foetal bovine serum, 100,000 units penicillin and 10.0 mg streptomycin per litre of medium and buffered with 15 mM Hepes and 23.6 mM NaHCO3, pH 7.30.

149

Bacillus cereus (ATCC No. 14579) and Staphylococcus aureus (ATCC No. 29213) and the Gramnegative bacteria Pseudomonas aeruginosa (ATCC No. 27853) and Escherichia coli (ATCC No. 25922). Minimum inhibitory concentrations (MIC) were determined using the microbroth dilution technique (Sahm and Washington, 1991). Dilutions of the oils were prepared in cation-adjusted Mueller Hilton Broth (CAMHB) beginning with 50 ll of 1% w/w solutions of essential oils in DMSO plus 50 ll CAMHB. The oil solutions were serially diluted (1:1) in CAMHB in 96-well plates. Organisms at a concentration of approximately 1.5 · 108 colony forming units (CFU)/mL were added to each well. Plates were incubated at 37
C for 24 h; the final minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive antibiotic control while DMSO was used as a negative control.

Cytotoxicity screening Hep G2 cells were plated into 96-well cell culture plates at 1.8 · 104 cells per well; Hs 578T cells at 1.0 · 105 cells per well and PC-3 cells at 1.9 · 104 cells per well. The volume in each well was 100 lL for all cell types. After 48 h, supernatant fluid was removed by suction and replaced with 100 lL growth medium containing either 2.5 or 1.0 lL of DMSO solution of oils or compounds (1% w/w in DMSO), giving a final concentration of 250 or 100 lg/mL, respectively, for each oil or compound. Hep G2 and Hs 578T cells were tested with final concentrations at 250 lg/mL and PC-3 at final concentration of 100 lg/mL. Solutions were added to wells in four replicates. Medium controls and DMSO controls (25 or 10 lL DMSO/mL) were used. Tingenone (250 or 100 lg/mL) was used as a positive control (Setzer et al., 1998). After the addition of compounds, plates were incubated for 48 h at 37
C; medium was then removed by suction, and 100 lL of fresh medium was added to each well. In order to establish percent kill rates, the CellTiter 96 AQueous Non-Radioactive Cell Proliferation assay was performed (Promega Technical Bulletin, 1996). After colorimetric readings were recorded (using a Molecular Devices SpectraMAX Plus microplate reader, 490 nm), average absorbances, standard deviations and percent kill ratios (% killcompd/% killDMSO) were calculated.

Antibacterial screening Both essential oils were screened for antimicrobial activities against the Gram-positive bacteria of

Results and discussion Chemical analysis of the components of the oils from the leaves and fruits of E. uniflora resulted in the identification of 36 and 32 components, respectively (Table 1). In both essential oils, sesquiterpenoids were the most abundant class of compounds, with the oxygenated derivatives being dominant. Monoterpene hydrocarbons, as usual, were in minor quantities (0.3–6.2%), while there was a general absence of their oxygenated counterparts. The major components of the leaf oil were curzerene (19.7%), selina-1,3,7(11)-trien-8-one (17.8%), atractylone (16.9%) and furanodiene (9.6%), while those of the fruits were germacrone (27.5%), selina-1,3,7(11)-trien-8-one (19.2%), curzerene (11.3%) and oxidoselina-1,3,7(11)-trien8-one (11.0%). The abundance of curzerene, atractylone and germacrone in the essential oil is noteworthy and a marked deviation from previous investigations, in which selina-1,3,7(11)-trien-8-one, oxidoselina1,3,7(11)-trien-8-one, nerolidol, pulegone, limonene a- and b-selinene, and carvone were reported as constituents occurring in the highest proportions (de Morais et al., 1996; Henriques et al., 1993; Weyerstahl et al., 1988). Also, compounds such as pulegone, a-selinene, carvone and nerolidol, that are characteristic components of the said reports, were not identified in our oil samples. However, we could detect limonene and b-selinene in insignificant amounts. Concerning the fruit volatile oil, furanodiene was not identified from this study

150 Table 1 RIa 854 938 992 1021 1024 1036 1046 1095 1337 1391 1411 1418 1430 1434 1439 1454 1461 1477d 1480 1487 1493e 1497 1497 1499 1513 1519f 1526 1541 1542 1557 1576 1584 1594 1601 1614 1625 1639 1652 1662 1663 1665 1687 1690 1727

I.A. Ogunwande et al. Constituents of the essential oils from the leaves and fruits of Eugenia uniflora Compoundsb,c

Percentage composition

trans-2-Hexenal a-Pinene Myrcene p-Cymene Limonene cis-Ocimene trans-Ocimene Terpinolene d-Elemene b-Elemene a-Gurjunene b-Caryophyllene b-Gurjunene (=Calarene) c-Elemene Aromadendrene a-Humulene Alloaromadendrene c-Selinene Germacrene D b-Selinene d-Selinene Ledene (=Viridiflorene) Bicyclogermacrene Curzerene c-Cadinene b-Cadinene d-Cadinene 4,7(11)-Selinadiene (=4,7(11)-Eudesmadiene) 3,7(11)-Selinadiene (=3,7(11)-Eudesmadiene) Germacrene B Spathulenol C15H24O Guaiol trans-b-Elemenone C15H26O Selina-1,3,7(11)-trien-8-one s-Cadinol Atractylone C15H26O C14H16O2 C14H16O2 Furanodiene Germacrone Oxidoselina-1,3,7(11)-trien-8-one

Leaves

Fruits

Tr Tr Tr Tr Tr 0.1 0.2 Tr 0.4 2.7 Tr 3.9 Tr 1.0 0.1 0.3 0.2 0.3 1.2 0.5 Nd Nd 2.4 19.7 Nd 0.5 0.4 0.2 0.3 5.8 1.5 1.0 1.4 0.9 Nd 17.8 Nd 16.9 Nd 1.1 1.0 9.6 2.6 5.9

Nd 0.1 0.5 Nd Nd 1.6 3.9 0.1 0.2 1.5 Nd 2.2 Nd 0.7 Tr 0.3 0.1 0.9 0.9 0.6 0.3 1.6 Nd 11.3 0.2 Tr 0.3 0.8 0.4 4.0 0.4 0.8 0.7 1.6 0.5 19.2 0.6 4.5 0.6 Nd Nd Nd 27.5 11.0

Nd, not detected; Tr, trace < 0.1%. a Retention indices on HP-5ms capillary coated column. b Order of elution on HP-5ms capillary coated column. c Identified by comparison of the mass spectral and retention index data. d RI value from Roussis et al., 2000 (see references). e RI value from Pala-Paul et al., 2001 (see references). f RI value from Skaltsa et al., 2003 (see references).

contrary to previous studies. These results may support the delineation of a new chemotype of the plant.

The fruit essential oils exhibited a strong antibacterial effect against Staphylococcus aureus, while the leaves oil showed a strong inhibition to

Studies on the essential oils composition, antibacterial and cytotoxicity of Eugenia uniflora L. Table 2

151

Antibacterial activities of the volatile oils (MIC lg/mL)

Sample

B. c

S. a

E. c

P. a

Fruit oil Leaf oil Gentamicin sulphate

625 39 1.22

39 156 0.61

625 625 2.44

625 625 1.22

B. c, Bacillus cereus (ATCC No. 14579); S. a, Staphylococcus aureus (ATCC No. 29213); E. c, Escherichia coli (ATCC No. 25922); P. a, Pseudomonas aeruginosa (ATCC No. 27853).

Table 3

Cytotoxic potentials of the essential oilsa

Sample

PC-3b

Hep G2c

Hs 578Td

Fruit oil Leaf oil

99.36 (0.11) 99.55 (0.16)

99.71 (0.29) 99.96 (0.04)

100 100

a b c d

% kill at tested concentrations (standard deviations in parentheses). Human prostate tumour cells (concentration tested 100 lg/mL). Human liver tumour cells (concentration tested 250 lg/mL). Human breast (ductal) tumour cells (concentration tested 250 lg/mL).

the growth of Bacillus cereus, with an MIC of 39 lg/ mL (Table 2). On the other hand, both volatile oils displayed an excellent cytotoxic action towards the human tumour cell lines of PC-3 and Hep G2, while completely inhibiting the growth of Hs 578T (see Table 3). Many claims are made regarding essential oils and their pharmacological and/or medicinal importance. Whilst essential oils might not be the ‘wonder drugs’ for the treatment of human cancers, the oils tested certainly deserve some further investigation. Although all in vitro experiments hold limitations with regards to possible in vivo efficacy, the results of this study are very promising with regards to possible anti-neoplastic chemotherapy and form a very sound basis for future research. This research will involve the isolation and characterization of compounds responsible for the observed biological activities of these essential oils.

Acknowledgments We are grateful to Mrs. Muslimat Bukari for her assistance in sample collection and preparation. Thanks are also due to Ms. A.K. Boehme and Ms. A. Bansal for assistance with the bioactivity screening. W.N.S. acknowledge generous financial support from an anonymous private donor.

References Adams RP. Identification of essential oil components by gas chromatography/mass spectrometry. Illinois: Allured Publishing Corporation Carol Stream; 2001.

Adebajo AC, Oloke KJ, Aladesanmi AJ. Antimicrobial activities and microbial transformations of volatiles oils of Eugenia uniflora. Fitoter 1989;50:451–5. Barbara CL, Sorensen J, Veal L. Vitex agnus castus essential oil and menopausal balance: a self care survey. Int J Aroma 2003;13:157–215. Baylac S, Racine P. Inhibition of human leukocytes elastase by natural fragrant extracts of aromatic plants. Int J Aroma 2004;14:179–82. British Pharmacopoeia, II, P.A. vol. 109, H.M. Stationary Office: London; 1980. Crowell PL. Prevention and therapy of cancer by dietary monoterpenes. Nutrition 1999;129:775–8. de Morais SM, Craveiro AA, Iracema M, Machado L, Alencar JW, Matos FJA. Volatile constituents of Eugenia uniflora leaf oil from Northeastern Brazil. J Essent Oil Res 1996;8: 449–51. Gbolade AA, Adebajo AC. Fumigant effects of some volatiles ions on fencundity and adult emergence of Callosobruchus maculatus F. Insect Sci Appl 1993;14:631. Hackett AJ, Smith HS, Springer EL, Owens RB, Nelson-Rees WA, Riggs JL, et al. Two syngeneic cell lines from human breast tissues: the aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines. J Nat Cancer Inst 1977;58:1795–806. Henriques AT, Sobral ME, Caudaro AD, Schapoval EES, Bassani VL, Lamaty G, et al. Aromatic plants from Brazil. II. The chemical composition of some Eugenia essential oils. J Essent Oil 1993;5:501–5. Iraj R, Mirmostafa SA. Bacterial susceptibility to and chemical composition of essential oils from Thymus kotschyamus and Thymus persicus. J Agric Food Chem 2003;51:2200–5. Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones L. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest Urol 1979;17:16–23. Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980;209:497–9. Lee M-H, Nishimoto S, Yang L-L, Yen K-Y, Hatano M, Yoshida T, et al. Two macrocyclic hydrolysable tannin dimers from Eugenia uniflora. Phytochemistry 1997;44:1343. Legault J, Dahl W, Debiton E, Pichette A, Madelmont J-C. Antitumor activity of balsam fir oil: production of reactive

152 oxygen species induced by a-humulene as possible mechanism of action. Planta Med 2003;69:402–7. Matsumura T, Kasai M, Hayashi T, Arisawa M, Momose Y, Arai I, Amagaya S, Komatsu Y. a-Glucosidase inhibitors from Paraguayan natural medicine, Nangapiry, the leaves of Eugenia uniflora. Pharm Biol 2000;38:302–7. Oladimeji FA, Orafidiya LO, Okeke IN. Physical properties and antimicrobial activities of essential oil of Lippia multiflora Moldenke. Int J Aroma 2004;14:162–8. Pala-Paul J, Velasco-Negueruela A, Perez-Alonso MJ, Sanz J. Analysis of the volatile components of Argyranthemum adauctum (Link). Humphries by gas chromatography-mass spectrometry. J Chromatogr A 2001;923:295–8. Piccaglia R, Marotti M, Galletti GC. Characterization of essential oil from a Satureja montana L., a chemotype grown in northern Italy. J Essent Oil Res 1991;3:147–52. Prichard AJN. The use of essential oils to treat snoring. Phytother Res 2004;18:696–9. Promega Technical Bulletin # 245. CellTiter 96R AQueous one solution cell proliferation assay. Madison, Wisconsin, USA: Promega Corporation; 1996. Roussis V, Tsoukatou M, Petrakis PV, Chinou I, Skoula M, Harbone JF. Volatile constituents of four Helichrysum species growing in Greece. Bio Syst Eco 2000;28:163–75.

I.A. Ogunwande et al. Sahm DH, Washington JA. Antibacterial susceptibility tests dilution methods. In: Balows A, Hausler WJ, Herrmann KL, Isenberg HD, Shamody HJ, editors. Manual of clinical microbiology. Washington DC, USA: American Society for Microbiology; 1991. Setzer WN, Setzer MC, Hopper AL, Moriarity DM, Lehrman GK, Niekamp KL, et al. The cytotoxic activity of a Salacia liana species from Monteverde, Costa Rica, is due to a high concentration of tingenone. Planta Med 1998;64: 583. Shu CK, Lawrence BM. Reasons for the variation in composition of some commercial essential oils. In: Risch SJ, Ho CT, editors. Spices, flavour chemistry and antioxidant properties. ACS symposium series, vol. 660. Washington, DC, USA: American Chemical Society; 1977. p. 138. Skaltsa HD, Demetzos C, Lazari D, Sokovic M. Essential oil analysis and antimicrobial activity of eight Stachys species from Greece. Phytochemistry 2003;64:743–52. Takahashi Y, Inaba N, Kuwahara S, Kuri W. Antioxidative effect of citrus essential oil components on human low-density lipoprotein in vitro. Biosci Biotechnol Biochem 2003;67: 195–7. Weyerstahl P, Marschall-Weyerstahl H, Christiansen C, Oguntimein BO, Adeoye AO. Volatile constituents of Eugenia uniflora leaf oil. Planta Med 1988;54:546–9.