Eugenia umbelliflora Fruits and Their Antibacterial Activity

Article pubs.acs.org/JAFC

Macrocarpal-like Compounds from Eugenia umbelliflora Fruits and Their Antibacterial Activity Larissa Gabriela Faqueti,† Ingrid Vicente Farias,† Elem Cristina Sabedot,† Franco Delle Monache,† Arturo San Feliciano,‡ Ivânia Teresinha Albrecht Schuquel,§ Valdir Cechinel-Filho,† Alexandre Bella Cruz,† and Christiane Meyre-Silva*,† †

Centro de Ciências da Saúde, Universidade do Vale do Itajaı ́ (UNIVALI), Itajaı ́, Santa Catarina State Brazil Department of Pharmaceutical Chemistry, Faculty of Pharmacy−CIETUS, University of Salamanca, 37007, Salamanca, Spain § Departamento de Quı ́mica, Universidade Estadual de Maringá (UEM), Maringá, Paraná State Brazil

Downloaded by 137.207.120.173 on September 14, 2015 | http://pubs.acs.org Publication Date (Web): September 14, 2015 | doi: 10.1021/acs.jafc.5b03562

‡

ABSTRACT: Certain members of the genus Eugenia are used as foods. One of these species is Eugenia umbelliflora, which is used for its fruits. The aim of the study was to isolate the constituents of the CH2Cl2 fraction obtained from E. umbelliflora O. Berg (Myrtaceae) and also to evaluate its antimicrobial properties. Two new meroterpenoids, eugenial C (3) and eugenial D (4) were isolated from the unripe fruits of E. umbellif lora and their structures established mainly by extensive NMR spectroscopy. In previous studies, the CH2Cl2 extract showed significant antibacterial activity, which can be attributed to meroterpenoids isolated in this study. The compounds eugenials C and D exhibited potent activity against Bacillus subtilis and Staphylococcus aureus and different strains of MRSA and activity similar to those of the antibiotics used in antimicrobial therapies. KEYWORDS: Eugenia umbelliflora, Myrtaceae, antibacterial activity, phloroglucinol-sesquiterpene, eugenial C, eugenial D

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INTRODUCTION Many species of Eugenia (Myrtaceae) are appreciated for their edible fruits.1 These include Eugenia uniflora L., popularly known as “cherry tree”, Eugenia edulis (O. Berg) Benth. & Hook. ex Griseb., known as jabuticaba, and the species studied here, Eugenia umbelliflora O. Berg., known as baguaçu.2 These species have been used in folk medicine to treat wounds, infections, and inflammation.1,3−6 There are reports in the literature of bactericidal activity of other species in this genus, namely, E. unif lora7 and E. caryophyllata Thunb.8 During a screening for bioactive compounds from plants in the Brazilian state of Santa Catarina, Machado et al.9 found high levels of antimicrobial activity in the CH2Cl2 fraction of methanolic fruit extract of E. umbellif lora. From the ripe fruits, Kuskoskis et al.10 reported the isolation of six anthocyanins, whereas Faqueti et al.11 reported the isolation from unripe fruits of a pair of isomeric meroterpenoids, named eugenial A (1) and eugenial B (2) (Figure 1). Although in this study both isomers could be separated, neither of them showed the antimicrobial activity previously observed in the crude extract.9,12 Therefore, less polar fractions were further processed to identify the lead compounds responsible for the activity found in the extract. This investigation focuses on the isolation of two new active compounds consisting of a phloroglucinol moiety coupled to a sesquiterpene, and on their antimicrobial evaluation.

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Figure 1. Structures of compounds 1−4 isolated from Eugenia umbellif lora fruits and of model compound macrocarpal C/G (5).13 spectrometer. HRESIMS were performed on an Agilent Technologies 6890N gas chromatograph connected to an MSD 5975 and a GCMSD ChemStation 61701DA. The column used was an Ultra-1 (J&W Scientific, Folsom, CA, USA; 25 m, 0.32 mm i.d.; film = 0.52 μm) with the following conditions: split injection, 0.5 mL of sample; flow rate, 1.0 mL/min (helium). The temperature program was as follows: initial temperature (iT), 150 °C (3 min); final temperature (fT), 280 °C (25 min); rate, 10 °C/min. NMR spectra were recorded on a Bruker DRX-

MATERIALS AND METHODS

General Experimental Procedures. Melting points were determined with an MQ APF-301 apparatus (Microquı ́mica, Palhoça, SC, Brazil) and are uncorrected. Optical rotations were measured using a digital polarimeter (341 PerkinElmer, Waltham, MA, USA) equipped with a sodium lamp; the samples for polarimetry were dissolved in CHCl3. IR spectra were recorded on an MB-100 Bomen © XXXX American Chemical Society

Received: April 30, 2015 Revised: August 21, 2015 Accepted: August 25, 2015

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DOI: 10.1021/acs.jafc.5b03562 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article


Journal of Agricultural and Food Chemistry 400 spectrometer (1H at 300 MHz), on an Avance III HD 500 MHz Bruker spectrometer (Bruker BioSpin, Rheinstetten, Germany), and/ or on a Mercury plus 300 MHz Varian spectrometer (Varian Inc., Palo Alto, CA, USA). CDCl3 was used as solvent, and the chemical shifts are given in parts per million referring to the residual nondeuterated solvent signal (CDCl3 7.27 and 77.0 ppm for 1H and 13C, respectively). Column chromatography was performed with Si gel (70−230 mesh) from Merck (Darmstadt, Germany). TLC analysis was carried out using Si gel GF-254, 0.2 mm precoated plates (Merck): the spots were visualized either using a UV lamp at 254 nm or by spraying with 2% sulfuric acid in ethanol. Plant Material. The unripe fruits of E. umbellif lora were collected in June 2008 in Itapema, Santa Catarina, Brazil, and identified by Prof. Msc. Oscar Benigno Iza (Universidade do Vale do Itajaı ́, Itajaı ́-SC). A voucher specimen (VC Filho 50) was deposited at the Barbosa Rodrigues Herbarium, Itajaı ́, Santa Catarina, Brazil. Extraction and Isolation. The unripe fruits, air-dried and cut into small pieces (310.45 g), were extracted twice with methanol (MeOH) at room temperature over a period of 7 days. The combined MeOH extracts were evaporated under reduced pressure with a rotary evaporator at 40 °C to obtain a brown gummy residue, which was suspended in a solution of MeOH/H2O (4:1) and successively partitioned with n-hexane (300 mL), CH2Cl2 (300 mL), and ethyl acetate (300 mL) to give three different nonpolar extracts. The residue that remained after partitioning was dried under reduced pressure to give the residual extract. Part of the CH2Cl2 extract (8.7 g) was subjected to silica gel 60 (Merck) open column chromatography (4 × 50 cm) and eluted with 400 mL each of a gradient of n-hexane/ CH2Cl2 in ratios to 100:0, 90:10, 80:20, 70:30, 60:40, and 50:50, respectively. On the basis of similar Rf values on TLC developed with the eluent n-hexane/CH2Cl2 (3:7) and sprayed with 10% sulfuric acid, six fractions (C1−C6) were grouped. Fraction C3 (0.8 g) was further purified by silica gel 60 open column chromatography (1.5 × 25 cm) and eluted with 200 mL each of mixtures of n-hexane/CH2Cl2 in ratios 100:0, 90:10, and 80:20 and finally purified by centrifugally accelerated preparative TLC on a Chromatotron (Harrison Research, Palo Alto, CA, USA) on 1 mm plates with hexane/CH2Cl2 (9:1), yielding 42 mg of compound 1 and 82 mg of compound 2.8 Fraction C4 (1.2 g) was further purified by silica gel 60 open column chromatography (2 × 30 cm) and eluted with 300 mL each of a mixture of n-hexane/CH2Cl2 in ratios of 80:20, 70:30, and 60:40, respectively, yielding 125 mg of a white precipitate, which was eugenial C (3). Fraction C5 (1.6 g) was purified by centrifugally accelerated preparative TLC and eluted with 500 mL of a gradient of n-hexane/ CH2Cl2 (70:30) to give 45 mg of eugenial D (4). Eugenial C (3): amorphous white solid; mp 136−138 °C; [α]D −7 (c 0.13, CHCl3); IR (KBr) 3503, 2960, 2884, 1621, 1458, 1380 cm−1; NMR data, see Table 1; EIMS (%) 440 (2), M•+ 237 (60) C12H13O5, phloroglucinol, 219 (18), 203 (100) C15H23, sesquiterpene; 161 (12), 149 (7), 147 (40), 129 (20), 105 (35). HRESIMS, found 440.2781 (calcd 440.2793 for C27H36O5). Eugenial D (4): amorphous white solid; mp 162−165 °C; [α]D −22 (c 0.22, CHCl3); IR (KBr) 3505, 2960, 2875, 1625, 1437, 1380 cm−1; NMR data, see Table 2; EIMS m/z (%) 440 (30), M•+ 237 (100) C12H13O5, phloroglucinol, 219 (21), 203 (95) C15H23, sesquiterpene, 161 (25), 149 (24), 147 (55), 129 (30), 105 (40); HRESIMS, found 440.2793 (calcd 440.2787 for C27H36O5). Microorganisms, Media, and Inocula. For the antimicrobial evaluation, strains of the American Type Culture Collection (ATCC), Rockville, MD, USA, were used, as follows: bacteria, Bacillus subtilis ATCC 23858, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 6538P; and yeast Candida albicans ATCC 10231. The methicillin-resistant Staphylococcus aureus (MRSA) strains (Sa3, Sa4, Sa5, Sa6, Sa8) were obtained from clinical material collected at the Hospital and Maternity Marieta Konder Bornhausen in Itajaı ́, Santa Catarina, Brazil. The strains were deposited at the Laboratory of Research in Microbiology Univali in Itajaı ́, Santa Catarina, Brazil. Quantitative Antimicrobial Evaluation. The minimum inhibitory concentration (MIC) of each compound was determined using

Table 1. NMR Data of Eugenial A (1),11 Eugenial C (3), and Macrocarpal C/G (5)16 1a

5b

3c

position

δC

δC

δC

1 2 2-OH 3 4 4-OH 5 6 6-OH 7

100.5 161.8

106.77 171.70

104.4 s 162.1 s

103.9 168.3

106.77 171.17

103.3 s 168.0 s

103.8 171.9

106.77 170.05

104.1 s 172.7 s

20.6

39.16

32.9 t

8 9 10 11 12 13 1′ 2′

191.8 206.7 45.9 17.9 14.0 23.0 78.3 130.1

193.45 193.29 36.48 28.71 24.47 52.92 29.17

50.8 d 26.0 t

3′

133.1

37.91

40.1 t

4′ 5′ 6′ 7′ 8′

38.7 27.4 33.3 27.6 33.1

50.33 52.15 28.89 29.96 27.34

47.0 53.7 27.7 27.5 25.5

9′

19.8

41.06

39.3 t

10′ 11′ 12′ 13′ 14′

19.6

156.89 21.51 18.04 29.96 106.77

15′

δH (J in Hz)

5.77 brs

14.42 s

25.48

192.1 206.9 46.1 17.8 13.8

154.8 20.1 16.6 28.9 105.5

d s t t q

s d d d t

s s q q t

20.4 q

15.49 s 2.28 d (14.8) 2.72 d (14.8) 10.10 s 3.12 t (7.5) 1.74 sex (7.5) 1.02 t (7.5) 2.45 1.76 1.92 1.46 1.64

m m m m m

1.22 0.62 0.79 1.01 2.06 2.03 2.45

dd (10.5, 10.5) dd (10.5, 10.5) ddd (10.5, 10.5, 6.5) m m dd (12.5, 12.5) m

1.03 1.12 4.76 4.70 0.92

s s brs brs s

a

In CDCl3 at 75.5 MHz (13C). bIn CD3OD at 67.5 MHz (13C). cIn CDCl3 at 500 MHz (1H) and 75.5 MHz (13C). Multiplicity for 13C assigned by DEPT method: s = C, d = CH, t = CH2, and q = CH3. broth microdilution techniques, following the guidelines of CLSI13 M07-A8 for bacteria and CLSI (formerly NCCLS) M27-A2 for yeasts.14 Stock solutions of compounds diluted in DMSO were added to each medium to give serial 2-fold dilutions, resulting in concentrations ranging from 1000 to 62.5 μg/mL and also from 100 to 0.19 μg/mL. Inocula of 100 μL (5 × 105 CFU/mL for bacteria and 103−104 CFU/mL for yeasts) were added to Mueller−Hinton broth medium (Merck) for bacteria and RPMI-1640 broth medium (Gibco, Grand Island, NY, USA) for yeast. Vancomycin hydrochloride (Sigma, St. Louis, MO, USA) and amphotericin B (Sigma) were used as positive controls. Plates were incubated for 24 h at 35 °C for bacteria and at 30 °C for yeasts. MIC was defined as the lowest concentration of compound capable of causing total inhibition of the growth of microorganism compared with control. All of the antimicrobial assays were tested in duplicate. Statistical Analysis. The data are reported as mean ± standard error of the mean (SEM) and were compared using one-way analysis of variance (ANOVA), followed by Dunnett’s pairwise test, and p values 1000 >1000

>1000 >1000 0.78

a

Bs, Bacillus subtilis; Sa, Staphylococcus aureus; Sa3, Sa4, Sa5, Sa6, and Sa8, different strains of methicillin-resistant S. aureus; Ec, Escherichia coli; Ca, Candida albicans. D


Article


Journal of Agricultural and Food Chemistry

(7) Auricchio, M.; Bacchi, E. M. Folhas de Eugenia unif lora L. (pitanga): propriedades farmacobotânicas, quı ́micas e farmacológicas [Eugenia uniflora L. “Brazilian cherry” leaves: pharmacobotanical, chemical and pharmacological properties]. Rev. Inst. Adolfo Lutz 2003, 62, 55−61. (8) Oussalah, M.; Caillet, S.; Saucier, L.; Lacroix, M. Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control 2007, 18, 414−420. (9) Machado, K. E.; Cechinel Filho, V.; Tessarolo, M. L.; MeyreSilva, C.; Bella Cruz, A. Potent antibacterial activity of Eugenia umbellif lora. Pharm. Biol. 2005, 43, 636−639. (10) Kuskoski, E. M.; Vega, J. M.; Rios, J. J.; Fett, R.; Troncoso, A. M.; Asuero, A. G. Characterization of anthocyanins from the fruits of baguaçu (Eugenia umbellif lora Berg). J. Agric. Food Chem. 2003, 51, 5450−5454. (11) Faqueti, L. G.; Maes Petri, C.; Meyre-Silva, C.; Machado, K. E.; Bella Cruz, A.; Garcia, P. A.; Cechinel Filho, V.; San Feliciano, A.; Delle Monache, F. Euglobal-like compounds from the genus Eugenia. Nat. Prod. Res. 2013, 27, 28−31. (12) Machado, K. E.; Cechinel-Filho, V.; Bella Cruz, R. C.; MeyreSilva, C.; Bella Cruz, A. Antifungal activity of Eugenia umbellif lora against dermatophytes. Nat. Prod. Commun. 2009, 4, 1181−1184. (13) CLSI − Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M07-A8; CLSI: Wayne, PA, USA, 2009. (14) NCCLS. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved Standard, 2nd ed.; NCCLS document M27-A2; NCCLS: Wayne, PA, USA, 2002. (15) Singh, I. P.; Bharate, S. B. Phloroglucinol compounds of natural origin: synthetic aspects. Nat. Prod. Rep. 2006, 23, 558−591. (16) Yamakoshi, Y.; Murata, M.; Shimizu, A.; Homma, S. Isolation and characterization of macrocarpals B−G antibacterial compounds from Eucalyptus macrocarpa. Biosci., Biotechnol., Biochem. 1992, 56, 1570−1576. (17) Nishizawa, M.; Emura, M.; Kan, Y.; Yamada, H.; Ogawa, K.; Hamanaka, N. HIV-RTase inhibitors of Eucalyptus globulus. Tetrahedron Lett. 1992, 33, 2983−2986. (18) Adio, A. M.; Paul, C.; Tesso, H.; Kloth, P.; König, W. A. Absolute configuration of helminthogermacrene. Tetrahedron: Asymmetry 2004, 15, 1631−1635. (19) Lewis, K. In search of natural substrates and inhibitors of MDR pumps. J. Mol. Microbiol. Biotechnol. 2001, 3, 247−254. (20) Tegos, G.; Stermitz, F. R.; Lomovskaya, O.; Lewis, K. Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrob. Agents Chemother. 2002, 46, 3133−3141. (21) Rudkin, J. K.; Edwards, A. M.; Bowden, M. G.; Brown, E. L.; Pozzi, C.; Waters, E. M.; Chan, W. C.; Williams, P.; O’Gara, J. P.; Massey, R. C. Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant Staphylococcus aureus by interfering with the agar quorum sensing system MRSA. J. Infect. Dis. 2012, 205, 798−806. (22) Rafii, F.; Sutherland, J. B.; Cerniglia, C. E. Effects of treatment with antimicrobial agents on the human colonic microflora. Ther. Clin. Risk. Manage. 2008, 4, 1343−1357.

presented antibacterial activity. The new compound, 3, also presented a similar antimicrobial profile. With regard to compounds 3 and 4, equivalent activity to the antibiotics commonly used in anti-infection therapy was found. Due to their superior activity against S. aureus (MIC 3.12 μg/ mL), 3 and 4 were also submitted to the test against different strains of MRSA. The results showed that the antibacterial activities of these compounds were similar to that of the standard strain of the bacteria, demonstrating their potential against MRSA (MIC = 0.39−3.12 μg/mL). Given that nowadays MRSA is a major cause of nosocomial infections worldwide and may be related to exposure to broadspectrum antibiotics, the discovery of a compound that can be specific to a single bacterium, as described in this study, can contribute to treatment,21 as it minimizes interference with the host microbial flora.22 In conclusion, this study allowed the isolation of two previously unknown phloroglucinols from the fruits of E. umbelliflora that showed high antibacterial activity mainly against MRSA standardized and clinical strains, similar to that of antibiotics in clinical use.

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AUTHOR INFORMATION

Corresponding Author

*(C.M.-S.) Phone: +55 47 33417664. Fax: +55 47 33417744. E-mail: [email protected]. Funding

We are grateful to CNPq, FAPESC-SC, and ProPPEC/ UNIVALI for providing financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Msc Oscar Benigno Iza (UNIVALI) for the classification and collection of plant material, Dr. Luis M. Peñ a-Rodrı ́guez and Fabiola Escalante-Erosa (Centro de Investigación Cientı ́fca de Yucatán-México) for the HRESIMS analysis, and Dr. Luiz Carlos Dias and Emıĺ io Júnior (UNICAMP) for the optical rotations.

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REFERENCES

(1) Lorenzi, H.; Matos, F. J. A. Plantas Medicinais no Brasil Nativas e Exóticas [Medicinal Plants in Native and Exotic Brazil]; São Paulo, Instituto Plantarum de Estudos da Flora Ltda: Nova Odessa, Brazil, 2002. (2) Mosimann, R. M. S.; Reis, A. Frutos nativos da Ilha de Santa Catarina, Florianópolis [Frutis natives from Santa Catarina island, Florianópolis]. Insula 1975, 8, 30−46. (3) Coelho de Souza, G.; Haas, A. P.S.; Von Poser, G. L.; Schapoval, E. E. S.; Elisabetsky, E. Ethnopharmacological studies of antimicrobial remedies in the south of Brazil. J. Ethnopharmacol. 2004, 90, 135−143. (4) Hussein, S. A. M.; Hashem, A. N. M.; Seliem, M. A.; Lindequist, U.; Nawwar, M. A. M. Polyoxygenated flavonoids from Eugenia edulis. Phytochemistry 2003, 64, 883−889. (5) Oliveira, R. N.; Camara, C. A. G. Estudo comparativo do óleo essencial de Eugenia punicifolia (HBK) DC. de diferentes localidades de Pernambuco [Comparative study of the essential oil of Eugenia punicifolia (HBK) DC. from different locations of Pernambuco]. Rev. Bras. Farmacogn. 2005, 15, 39−43. (6) Romagnolo, M. R.; Souza, M. C. O gênero Eugenia L. ́ de alagável do Alto Rio Paraná, Estados de (Myrtaceae) na planicie Mato Grosso do Sul e Paraná, Brasil [The genus Eugenia L. (Myrtaceae) on the Upper Paraná River floodplain, Mato Grosso do Sul and Paraná States, Brazil]. Acta Bot. Bras. 2006, 20, 529−548. E


Eugenia umbelliflora Fruits and Their Antibacterial Activity

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