PHYTOTHERAPY RESEARCH Phytother. Res. (2013) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.4987

SHORT COMMUNICATION

Quinolone Alkaloids from Fructus Euodiae Show Activity Against Methicillin-Resistant Staphylococcus aureus Xiaobei Pan, S. W. Annie Bligh and Eileen Smith* School of Human Sciences, Faculty of Life Sciences and Computing, London Metropolitan University, 166-220 Holloway Road, London N7 8DB, UK

Fructus Euodiae is used in Traditional Chinese Medicine to treat infection. In this study, four of the quinolone alkaloids isolated from Fructus Euodiae showed activity against methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) were 8–128 mg/mL, which were equivalent to or lower than the control antibiotics, oxacillin, erythromycin and tetracycline (MIC ≥128 mg/mL). Among these isolated quinolone alkaloids, evocarpine with a 13 carbon alkenyl chain substituent at position-2 showed the best activity against MRSA. This study has demonstrated the potential of quinolone alkaloids from Fructus Euodiae as antiMRSA compounds and supports the traditional use of the fruit as a treatment for bacterial infections. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: Evocarpine; MRSA; antibacterial; minimum inhibitory concentration; MIC.

INTRODUCTION Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen which causes skin infections, including deep-skin abscesses, pneumonia bacteraemia and endocarditis. Both healthcare-associated and community-associated MRSA infections are becoming a serious challenge for treatment with antibiotics (Gould et al., 2012); therefore, new agents are needed to treat MRSA associated infections. ‘Fructus Euodiae’ (Chinese Pharmacopoeia 2010) and ‘Fructus Euodiae’ (CP 2005) both refer to the fruits of Tetradium ruticarpum (A. Jussieu) T.G. Hartley (formerly Evodia rutaecarpa (A. Juss.) Benth., Evodia ruticarpa var. bodinieri (Dode) C.C. Huang and Evodia ruticarpa var. officinalis (Dode) C.C. Huang. These are all now considered synonyms of one species. The dry, nearly ripe fruits of Tetradium ruticarpum are known as ‘Wu zhuyu’ in Chinese. This plant is used in Traditional Chinese Medicine to treat bacterial infection and inflammation-related disorders such as eczema and ulcers (King et al., 1980; Yarosh et al., 2006). Several active chemical constituents have been isolated from this plant including alkaloids, essential oils, limonoids, carboxylic acids and flavonoids (Wang and Liang, 2004). The main constituents isolated from Fructus Euodiae are alkaloids, including quinazolinocarboline, quinolone and indole alkaloids (Liu et al., 2005; Tang et al., 1996; Zuo et al., 2000). The inhibitory activity of quinolone alkaloids from Fructus Euodiae against the fast-growing mycobacterial strains, Mycobacterium fortuitum, M. smegmatis and M. phlei with minimum inhibitory concentration (MIC) * Correspondence to: Eileen Smith, School of Human Sciences, Faculty of Life Sciences and Computing, London Metropolitan University, 166-220 Holloway Road, London N7 8DB, UK. E-mail: [email protected]

Copyright © 2013 John Wiley & Sons, Ltd.

values between 2 mg/mL and 32 mg/mL has been demonstrated (Adams et al., 2005). The activity of synthetic N-methyl-2-alkenyl-4-quinolones against MRSA has also been reported (Guzman et al., 2011). Here we demonstrate the activity of quinolone alkaloids isolated from Fructus Euodiae against MRSA.

MATERIALS AND METHODS Plant material. The fresh fruit of Tetradium ruticarpum was collected from Taining county, Fujian province, P. R. China in 1999, and was authenticated by Dr. Lihong Wu, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine (SUTCM), P.R. China. The fruit was sun-dried, and a voucher specimen was deposited in the herbarium of Institute of Chinese Materia Medica, SUTCM.

Extraction and isolation. The dry fruit was ground to a fine powder. The powder (100 g) was soaked in ethyl acetate (800 mL) for 30 min and then extracted by ultrasonification for 45 min. The procedure was repeated three times, and then the extract was filtered and combined. The solvent was removed by rotary evaporation under reduced pressure to give a brown crude residue 5.4 g, which was stored at 4  C. The residue was subjected to separation by high-performance liquid chromatography (HPLC) using a method adapted from the literature (Liu et al., 2005). HPLC was carried out on a Dionex Ultimate 3000 HPLC system (CA, USA) which consisted of an Ultimate 3000 pump, a ternary gradient unit, an autosampler, an AFC 3000 fraction collector and an UVD340 detector. A semi-prep Knauer C18 Received 11 January 2013 Revised 11 February 2013 Accepted 05 March 2013

X. PAN ET AL.

(7.8 mm x 300 mm, 7 mm) column was used for separation with a gradient mobile phase of methanol, acetonitrile and water. The flow rate was set at 4.5 mL/min. The detection wavelength was set at 240 nm. The isolated compounds were characterized by nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization (ESI) mass spectrometry. NMR spectra were obtained using a Bruker Avance 500 NMR Spectrometer (Bruker, Coventry, UK). TMS (tetramethylsilane) was used as a reference for all NMR measurements. Mass spectra were achieved on a Waters Q-Tof microTM mass spectrometer (Waters, MA, USA) via an ESI positive ion source.

Bacterial strains. The MRSA strain ATCC 33591 and standard S. aureus strain ATCC 25923 were obtained from Oxoid (Basingstoke, UK). Escherichia coli strain NCIMB 11190 was obtained from the National Collection of Industrial and Marine Bacteria (Aberdeen, UK). All strains were cultured on nutrient agar (Oxoid) and incubated overnight at 37  C prior to assay.

MIC assay. Strains were made up in 0.9% saline to an inoculum density of 5  105 cfu by comparison with a MacFarland standard. Tetracycline, norfloxacin, ciprofloxacin, erythromycin and oxacillin were obtained from Sigma-Aldrich (Dorset, UK). Test samples and antibiotics were dissolved in dimethyl sulfoxide (DMSO), and a DMSO control was included. MICs were determined in duplicate using a standard broth microdilution assay as previously described (Smith et al., 2005).

RESULTS AND DISCUSSION 0.5 g of the residue was run on HPLC, and six alkaloids were collected: 1 (12.6 mg), 2 (1.8 mg), 3 (24.6 mg), 4 (6.3 mg) 5 (1.8 mg) and 6 (2.5 mg). The structures of the six quinolone alkaloids were determined by 1D (1H and 13C) and 2D (HMQC and HMBC) NMR spectroscopy and high-resolution ESI mass spectrometry. Comparison of the data with those reported in the relevant literature confirmed the compounds as quinolone alkaloids: 1, 2, 3 and 4 (Tang et al., 1996); 4 and 5 (Liu et al., 2005) (Fig. 1). Four of these isolated alkaloids (1, 2, 4 and 5) showed activity against both the MRSA and standard strains with MIC values of 8 – 128 mg/mL (Table 1). An Euodia crude extract has previously been shown to be active against S. aureus (Thuille et al., 2003), but here the activity of quinolone alkaloids isolated from Fructus Euodiae

Figure 1. Structures of isolated quinolone alkaloids. Copyright © 2013 John Wiley & Sons, Ltd.

Table 1. Minimum inhibitory concentrations (MICs) of four active quinolone alkaloids and standard antibiotics in mg/mL MIC in mg/mL Compounds

ATCC 33591 (MRSA)

ATCC 25923

1 2 4 5 Oxacillin Erythromycin Tetracycline Ciprofloxacin Norfloxacin

64 32 8 128 128 > 128 128 0.16 1

64 16 8 128 0.16 0.32 0.32 0.16 1.28

against MRSA was demonstrated for the first time. However, none of these compounds showed activity against the E.coli strain (MIC ≥ 128 mg/mL). The MRSA strain is highly resistant to oxacillin, erythromycin and tetracycline (MIC ≥ 128 mg/mL). Evocarpine (4) demonstrated the highest activity of 8 mg/mL, 16-fold more active than oxacillin against MRSA. Compounds 1 and 2 were also more active than oxacillin (two-fold and four-fold, respectively) and compound 5 showed the same activity as oxacillin (128 mg/mL). The two quinolone antibiotics ciprofloxacin and norfloxacin were active against both strains with MIC values against MRSA of 0.16 and 1 mg/mL respectively. Wube et al. (2011) reported an MIC of 2 mg/mL for evocarpine against M. smegmatis. A similar result for evocarpine (4) was obtained in this study with an MIC of 8 mg/mL against MRSA. Evocarpine, which showed the best anti-staphylococal activity, is a 1-methyl-2alkenyl-4(1H)-quinolone alkaloid with a 13-carbon alkenyl chain substituent at position-2. Many 1-methyl-2-alkenyl-4(1H)-quinolones have been synthesized and their anti-microbial activities evaluated in other studies (Guzman et al., 2011; Wube et al., 2011). Wube et al. (2011) demonstrated that a monounsaturated aliphatic side chain of 11–13 carbons at position-2 favours anti-mycobacterial activity of these alkaloids in vitro. Guzman et al. (2011) synthesized five 1-methyl-2-alkenyl-4(1H)-quinolones with an alkenyl chain length of 10 – 14 carbons which showed antiMRSA activity of 0.5 – 4 mg/mL. In this study, the best activity was also exhibited by compounds with a monounsaturated aliphatic side chain. Evocarpine (4) with a 13-carbon chain had the highest activity at 8 mg/mL and compound 2 with an 11-carbon chain was active at 32 mg/mL. Compound 3 showed modest activity at 64 mg/mL suggesting that a short saturated aliphatic side chain may contribute to antibacterial activity; however, compounds 3 and 6 with longer side chains (11 and 13 carbons respectively) had no activity. Compound 5 which had a 15-carbon chain with two double bonds was only active at 128 mg/mL. Fluoroquinolone antibiotics such as norfloxacin and ciprofloxacin exert their activity by inhibition of DNA gyrase and topoisomerase IV enzymes (Maxwell, 1999). Further studies will be carried out to investigate whether evocarpine and other quinolones isolated from Fructus Euodiae are inhibitors of DNA gyrase or topoisomerase IV. This study has demonstrated that Fructus Euodiae contains quinolone alkaloids with Phytother. Res. (2013)

ACTIVITY OF FRUCTUS EUODIAE AGAINST MRSA

activity against MRSA, with evocarpine being the most active alkaloid isolated. This result supports the traditional use of Fructus Euodiae to treat infections.

Conflict of Interest The authors have declared that there is no conflict of interest.

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Phytother. Res. (2013)

Quinolone alkaloids from Fructus Euodiae show activity against methicillin-resistant Staphylococcus aureus.

Fructus Euodiae is used in traditional Chinese medicine to treat infection. In this study, four of the quinolone alkaloids isolated from Fructus Euodi...
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