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Author's personal copy Fitoterapia 93 (2014) 62–66
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Antibacterial anthranilic acid derivatives from Geijera parviflora Qingyao Shou a, Linda K. Banbury a, Alan T. Maccarone c, Dane E. Renshaw a, Htwe Mon b, Stefani Griesser b, Hans J. Griesser b, Stephen J. Blanksby c, Joshua E. Smith a, Hans Wohlmuth d,⁎ a b c d
Southern Cross Plant Science, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia Division of Research, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia
a r t i c l e
i n f o
Article history: Received 13 October 2013 Accepted in revised form 8 December 2013 Available online 25 December 2013 Keywords: Geijera parviflora Rutaceae Anthranilic acid derivative Antibacterial activity
a b s t r a c t Five anthranilic acid derivatives, a mixture I of three new compounds 11′-hexadecenoylanthranilic acid (1), 9′-hexadecenoylanthranilic acid (2), and 7′-hexadecenoylanthranilic acid (3), as well as a new compound 9,12,15-octadecatrienoylanthranilic acid (4) together with a new natural product, hexadecanoylanthranilic acid (5), were isolated from Geijera parviflora Lindl. (Rutaceae). Their structures were elucidated by extensive spectroscopic measurements, and the positions of the double bonds in compounds 1–3 of the mixture I were determined by tandem mass spectrometry employing ozone-induced dissociation. The mixture I and compound 5 showed good antibacterial activity against several Gram-positive strains. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Geijera parviflora Lindl. (family Rutaceae), known locally as wilga or native willow, is an endemic Australian shrub or small tree growing to a height of 10 m. This species grows in mixed woodland communities and is widespread in inland parts of eastern Australia [1]. The aromatic leaves have been used by Indigenous Australians to alleviate pain including toothache and were also used for ceremonial purposes [2]. The species has previously been reported to contain coumarins [3,4], alkaloids [4], and essential oil [5] in its leaves and fruits, and our group has recently isolated two unusual new 11-C alkaloids from the leaves [6].
We now report the isolation and structural elucidation of five anthranilic acid derivatives: mixture I consisting of three new compounds, namely 11′-hexadecenoylanthranilic acid (1), 9′-hexadecenoylanthranilic acid (2), and 7′-hexadecenoylanthranilic acid (3), a new compound 9,12,15-octadecatrienoylanthranilic acid (4) and a new natural product hexadecanoylanthranilic acid (5) (Fig. 1). These compounds were tested to assess their antibacterial activity, with the mixture I and compound 5 showing good antibacterial activity against several Gram-positive strains. 2. Experimental 2.1. General
⁎ Corresponding author. Tel.: +61 2 66203000; fax: +61 2 66223459. E-mail addresses:
[email protected] (Q. Shou),
[email protected] (L.K. Banbury),
[email protected] (A.T. Maccarone),
[email protected] (D.E. Renshaw),
[email protected] (H. Mon),
[email protected] (S. Griesser),
[email protected] (H.J. Griesser),
[email protected] (S.J. Blanksby),
[email protected] (J.E. Smith),
[email protected] (H. Wohlmuth). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.008
UV spectra were obtained on a Hewlett Packard 8453 polarimeter at room temperature. IR spectra were acquired using a Bruker Vector 33 Spectrometer. High-resolution electrospray ionisation mass spectrometry (HRESI-MS) to obtain accurate mass measurements was carried out on a Bruker micrOTOF-Q instrument (Bremen, Germany) with a Bruker ESI source. NMR spectra were acquired on a Bruker
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Fig. 1. Structures of compounds 1–5.
AVANCE 500 MHz spectrometer with TMS as the internal standard. Column chromatography (CC) separations were carried out using silica gel (Silica-Amorphous, precipitated, 200–425 mesh, Sigma-Aldrich), and MCI gel CHP20P (Supelco, Bellafonte, PA, USA). Preparative HPLC was performed on a Gilson 322 system with a UV/Vis-155 detector and a FC204 fraction collector using a Phenomenex Luna 5 μm (150 × 21.2 mm i.d.) C-18 column. 2.2. Plant material The leaves of Geijera parviflora were collected near Lightning Ridge, New South Wales, Australia (29° 25′ S, 147° 59′ E) in December 2011 and authenticated by one of the authors (HW). A voucher specimen (PHARM110063) has been deposited in the Medicinal Plant Herbarium at Southern Cross University. 2.3. Extraction and isolation The powdered dried leaves of G. parviflora (2 kg) were extracted with 95% ethanol at room temperature. The ethanol extract was suspended in H2O and extracted using CHCl3 (3 × 1 L). The CHCl3 portion was evaporated under reduced pressure to afford a crude extract (167.5 g). The crude CHCl3 extract was subjected to MCI gel (CHP20P) CC, eluted with a gradient of MeOH/H2O (80:20–100:0) to give five fractions (A–E). Fraction E was subjected to a silica gel column, with hexane–EtOAc (8:1, 4:1) as eluent to give six subfractions (EI–EVI). Fraction EV (112 mg) was subjected to preparative HPLC [mobile phase: acetonitrile and H2O containing 0.05% TFA (0–5 min: 70% acetonitrile, 5–17 min: 70%–95% acetonitrile,
17–20 min: 95% acetonitrile); flow rate 20 mL/min] to give mixture I (5 mg) and compound 4 (7 mg). Fraction EVI (64 mg) was further separated by preparative HPLC [mobile phase: acetonitrile and H2O containing 0.05% TFA (0–5 min: 80% acetonitrile, 5–17 min: 80%–95% acetonitrile, 17–20 min: 95% acetonitrile); flow rate 20 mL/min] to give compound 5 (43 mg). 2.3.1. Mixture I (compounds 1–3) A light yellow oil; UV (MeOH) λmax (log ε) 215.0 (3.88), 253.0 (3.66), 297.0 (3.06) nm; IR (neat) νmax 2927.7, 2856.0, 1689.4, 1679.9, 1605.7, 1588.2, 1526.8, 1450.2, 1247.3, 759.3 cm−1; 1H NMR (500 MHz, CDCl3) δ 10.94 (1H, s, NH), 8.77 (1H, d, J = 8.6 Hz, H-3), 8.11 (1H, dd, J = 8.0, 1.6 Hz, H-6), 7.60 (1H, m, H-4), 7.11 (1H, m, H-5), 5.31-5.40 (2H, m, double bond protons in the hexadecenoyl groups), 2.45 (2H, t, J = 7.5 Hz, H-2′), 2.03 (4H, m, protons of CH2 near double bonds in the hexadecenoyl groups), 1.77 (2H, m, H-3′), 1.25– 1.45 (overlap), 0.91 (3H, m, H-16′); 13C NMR (125 MHz, CDCl3) δ 172.7 (C, C-1′), 171.5 (C, COOH), 142.7 (C, C-2), 135.8 (CH, C-4), 131.9 (CH, C-6), 129.8–130.6 (C, the double bond carbons in the hexadecenoyl groups), 122.8 (CH, C-5), 120.8 (CH, C-3), 113.9 (C, C-1), 39.0–39.1 (CH2, C-2′), 29.3–30.2 (the remaining CH2 in the hexadecenoyl groups), 27.3–27.6 (CH2, the allylic carbons in the hexadecenoyl group), 25.7–25.9 (CH2, C-3′), 22.7–23.2 (CH2, C-15′), 14.4–14.5 (CH3, C-16′); HRESI-MS m/z 396.2509 [M + Na]+ (Calc. 396.2515 for C25H35NNaO3); APCI-MS m/z 374.1 [M + H]+. 2.3.2. Compound (4) A yellow oil; UV (MeOH) λmax (log ε) 220.0 (4.08), 252.0 (3.84), 297.0 (3.34) nm; IR (neat) νmax 2927.3, 2855.6,
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1703.9, 1605.9, 1588.2, 1526.0, 1450.0, 1251.7, 759.8 cm−1; 1 H NMR (500 MHz, CDCl3) δ 10.97 (1H, s, NH), 8.77 (1H, d, J = 8.0 Hz, H-3), 8.10 (1H, dd, J = 8.0, 1.4 Hz, H-6), 7.59 (1H, m, H-4), 7.11 (1H, m, H-5), 5.29–5.47 (6H, m, H-9′, 10′, 12′, 13′, 15′, 16′), 2.81 (4H, m, H-11′, 14′), 2.10 (2H, m, H-17′), 2.08 (2H, m, H-8′), 0.98 (3H, t, J =7.6 Hz); 13C NMR (125 MHz, CDCl3) δ 172.9 (C, C-1′), δ 171.7 (C, COOH), δ 142.5 (C, C-2), 135.8 (CH, C-4), 132.4 (CH, C-6), 127.5, 128.1, 128.7, 128.7, 130.7, 132.1 (CH, C-9′, 10′, 12′, 13′, 15′, 16′), 122.8 (CH, C-5), 120.8 (CH, C-3), 114.5 (C, C-1), 39.1 (CH2, C-2′), 29.3–30.0 (CH2, C-4′-7′), 27.6 (CH2, C-8′), 26.1 (CH2, C-3′), 25.7, 25.8 (CH2, C-11′, 14′), 20.9 (CH2, C-17′), 14.7 (CH3, C-18′). HRESI-MS m/z 420.2517 [M + Na]+ (Calc. 420.2515 for C23H35NNaO3); APCI-MS m/z 398.1 [M + H]+. 2.3.3. Compound (5) A white solid; 1H NMR (500 MHz, CDCl3) δ 10.93 (1H, s, NH), 8.78 (1H, d, J = 8.5 Hz, H-3), 8.12 (1H, dd, J = 8.0, 1.6 Hz, H-6), 7.61 (1H, m, H-4), 7.12 (1H, m, H-5), 2.48 (2H, t, J = 7.6 Hz, H-2′), 1.77 (2H, m, H-3′), 1.21–1.45 (24H, overlap), 0.89 (3H, t, J = 6.5 Hz, H-16′); 13C NMR (125 MHz, CDCl3) δ 172.7 (C, C-1′), 171.6 (C, COOH), 142.5 (C, C-2), 135.9 (CH, C-4), 131.9 (CH, C-6), 122.7 (CH, C-5), 120.8 (CH, C-3), 113.9 (C, C-1), 39.0 (CH2, C-2′), 29.4-32.2 (CH2, C-4′-14′), 25.8 (CH2, C-3′), 22.9 (CH2, C-15′), 14.3 (CH3, C-16′). HRESI-MS m/z 398.2662 [M + Na]+ (Calc. 398.2671 for C23H35NNaO3); APCI-MS m/z 376.3 [M + H]+. 2.4. Ozone-induced dissociation of mixture I Tandem mass spectrometry was undertaken on a Thermo Fisher Scientific LTQ single stage linear ion-trap mass spectrometer (San Jose, CA, USA) that has been previously modified to allow ozone to enter the ion-trapping region [7]. [M-H]− ions were generated at m/z 372 by electrospray ionization of a methanolic solution of mixture I (25 μM). Collisioninduced dissociation mass spectra were obtained by massselection of the precursor ion (isolation width 1 Th) and activation (normalised collision energy 35 arbitrary units). Ozone-induced dissociation (OzID) was used to locate double bonds in the isomeric components of mixture I wherein ions were isolated in the presence of ozone for 10 s before being mass-analysed. High concentration ozone was generated online and entrained in the helium collision gas; the specific conditions have been recently described [8]. All tandem mass spectra represent an average of at least 100 individual scans. 2.5. Bioactivity Bacterial test strains [Staphylococcus aureus ATCC 29213, ATCC 25923, ATCC 43300, W/T (clinical isolate), 6538P; S. epidermidis ATCC 35984 (biofilm forming); S. haemolyticus IMVS 2782/97; S. saprophyticus IMVS 0684/85; Escherichia coli ATCC 25922; Pseudomonas aeruginosa ATCC 27853] from stock cultures preserved at −80 °C in the Microbiology Laboratory, University of South Australia, were utilized. All bacterial strains were grown on Columbia blood agar base (Oxoid CM331), supplemented with 2.5% (w/v) defibrinated horse blood. Tryptone soya broth (TSB) with 0.025 g/mL glucose prepared to manufacturer's specifications was used for the determination of minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC). Broth microdilution was performed in 96-well round bottom plates (Sarstedt, Technology Park, South Australia) according to the Clinical Laboratory Standards Institute's method M7-A7 with modification (TSB broth plus glucose in place of Mueller– Hinton broth) [9]. Serial 1-in-2 dilutions of the test compounds in TSB containing 2% DMSO were made in the wells, in a total of 50 μL. Bacterial strains were prepared by adding sterile saline to actively growing broth culture to achieve a turbidity equivalent to a standardized reading of 0.5 McFarland units representing 108 CFU/mL. These suspensions were diluted 1 in 100 in TSB broth, and 50 μL of inoculum was added to each well, delivering approximately 5 × 105 CFU in a total volume of 100 μL. The microtitre plate was incubated overnight in an incubator at 37 °C. The MIC was determined as the lowest concentration at which no growth was observed in the microdilution wells as detected by the unaided eye. Following determination of the MIC, a 10 μL aliquot from each of the wells at the concentration corresponding to the MIC and those concentrations above were transferred to 190 μL of TSB in a microtitre plate, which was incubated at 37 °C overnight. The MBC was determined as the lowest concentration at which no growth occurred and was confirmed by viable cell count on Columbia agar plates. 3. Results and discussion Chemical investigation of the ethanol extract of the leaves of Geijera parviflora led to the identification of five anthranilic acid derivatives (1–5). The structural elucidation of these compounds was performed by NMR analyses, including 1D and 2D NMR experiments, as well as collision- and ozoneinduced dissociation. Mixture I was obtained as a light yellow oil, its HRESI-MS exhibited an ion peak at m/z 396.2509, which suggested a molecular formula of C23H35NO3 ([M + Na]+ calcd 396.2515). The UV spectrum showed absorptions at 253 and 295 nm, indicating an anthranilic acid derivative [10]. The CID spectrum of the [M-H]− anion formed upon electrospray ionization of this compound(s) in negative ion mode is shown in Fig. 2(a). This spectrum reveals abundant product ions at m/z 136 and m/z 235 consistent with the presence of an anthranilic acid moiety and a monounsaturated 16-carbon acyl chain, respectively. In the 1H NMR spectrum, four vicinal aromatic protons were observed at δH 8.11 (dd, 1H, J = 8.0, 1.6), δH 8.77 (d, 1H, J = 8.6), δH 7.60 (m, 1H) and δH 7.11 (m, 1H); one proton at δH 10.94 indicated the presence of an amide group. Proton signals at δH 0.91 (m, 3H), δH 1.25–1.45, δH 1.77 (m, 2H), 2.03 (m, 4H), 2.45 (t, 2H, J = 7.5) as well as two protons at δH 5.31–5.40 suggested the presence of a long aliphatic chain with a double bond. The 13C NMR and HSQC spectrum showed signals for two carbonyls at δC 172.7, 171.5 and six aromatic carbons. In the HMBC spectrum, the correlations of δH 8.11 (H-6) with δC 171.5 (COOH), and of δH 10.94 (NH) with δC 172.7 (C-1′) and 120.8 (C-3) were observed. These spectroscopic data led to the establishment of a hexadecenoylanthranilic acid structure of mixture I, although the location of the double bond in the aliphatic chain remained unresolved. The location of the double bonds in the hexadecenoyl groups was examined by tandem mass spectrometry using
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b
Fig. 2. Negative ion ESI-MS of mixture I produced an abundant [M-H]− anion at m/z 372 that was then subjected to (a) collision-induced dissociation (CID, normalised collision energy 35 arbitrary units) and (b) ozone-induced dissociation (OzID, 10 s trapping time in the presence of ozone). The identities of major fragment ions are indicated.
the ozone-induced dissociation (OzID) protocol previously described [7]. A trapping time of 10 s was sufficient to produce unique spectral signatures indicating the presence of three monounsaturated isomers. The resulting OzID spectrum is shown in Fig. 2(b) and reveals peaks at m/z 318, m/z 290, and m/z 262 were observed, corresponding to neutral losses of 54, 82, and 110 Da and diagnostic for double bond positions at C-11′, C-9′ and C-7′, respectively [11]. The Z-form of the double bonds was established by considering the chemical shifts of the allylic carbons at δ 27.3–27.6 [12]. Thus, I was elucidated as a mixture of three isomers with different double bond positions, 11′-hexadecenoylanthranilic acid (1), 9′-hexadecenoylanthranilic acid (2), 7′-hexadecenoylanthranilic acid (3). We were unable to separate these isomers. Compound 4 was obtained as a light yellow oil, its HRESIMS exhibited an ion peak at m/z 420.2517, which suggested a molecular formula of C25H35NO3 ([M + Na]+ calcd 420.2515). The UV and 1H NMR spectra suggested an anthranilic acid derivative similar to mixture I but with an aliphatic chain of eighteen carbons. In the 1H NMR spectrum, six protons at δH 5.29–5.47 suggested the presence of three double bonds in the aliphatic chain; this was also supported by six olefinic carbons at δC 132.1, 130.7, 128.7, 128.7, 128.1 and 127.5. The HMBC correlations of the terminal methyl (δH 0.98, 3H) with δC 20.9 (C-17′) and δC 132.1 (C-16′) as well as the correlations from two methylenes (δH 2.81, 4H) to the six olefinic carbons suggested a 9,12,15- triene structure of the aliphatic chain. The Z-form of the three double bonds was established by considering the chemical shifts of the allylic carbons at 27.6 (C-8′), 25.7, 25.8 (C-11′, 14′), 20.9 (C-17′) consistent with the reported data of linolenic acid [13]. Based on these data, compound 4 was established as 9,12,15octadecatrienoylanthranilic acid. Compound 5 was a white solid, it showed a peak at m/z 398.2662 ([M + Na]+ calcd 398.2671), indicating a molecular
formula of C23H37NO3. Compared with mixture I, one degree of unsaturation less indicated a saturated hexadecanoyl group of the long aliphatic chain. This was also supported by the absence of the double bond protons at δH 5.31–5.40 in 1 H NMR spectrum. Thus, compound 5 was determined as hexadecanoylanthranilic acid, a new natural product which has previously been synthesized [14,15], but never before isolated from a natural source. Compound 4, with its polyunsaturated aliphatic chain, was found to be unstable and susceptible to oxidation; this prevented us from obtaining bioassay data for this compound. The mixture I and compound 5 were tested for antibacterial activity against Gram-positive and Gram-negative strains (Table 1). Both displayed anti-bactericidal activity against several Gram-positive strains, but not against the two Gramnegative strains. Compound 5 had low MIC values ranging from 0.66 to 2.97 μg/mL against six of eight Gram-positive strains, but was not active against S. aureus (ATCC 29213) or S. saprophyticus (IMVS 0684/85). The apparent selective activity of compound 5 is an interesting finding, and further studies are warranted to elucidate its pattern of activity against various strains and its mechanism of action. It is also worth noting that compound 5 and mixture I were active against a methicillin-resistant strain of Staphylococcus aureus (ATCC 43300) with MIC values of 1.30 and 5.94 μg/mL, respectively. Acknowledgments This work was supported by the Wound Management Innovation CRC (established and supported under the Australian Government's Cooperative Research Centres Program). The authors thank Graham Macfarlane of the School of Chemistry and Molecular Biosciences at the University of Queensland for determining the accurate mass of the compounds. S.J.B.
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Table 1 Anti bacterial activity of mixture I and 5a. Compounds
Mixture I
Compound 5
Staphylococcus aureus (ATCC 25923) Staphylococcus aureus (ATCC 29213) Staphylococcus aureus (ATCC 43300)b Staphylococcus epidermidis (ATCC 35984) Staphylococcus haemolyticus (IMVS 2782/97) Staphylococcus aureus W/T (clinical isolate) Staphylococcus aureus 6538P Staphylococcus saprophyticus (IMVS 0684/85) Pseudomonas aeruginosa (ATCC 27853) Escherichia coli (ATCC 25922)
11.87 (11.87) 23.75 (47.5) 5.94 (11.87) 5.94 (11.87) NT
2.97 (5.94) N190 (NA) 1.30 (2.70) 0.66 (1.30) 1.30 (2.60)
NT
1.30 (2.60)
NT NT
2.60 (5.30) NA
N95 (NA) NT
N190 (NA) N190 (NA)
a Values shown are MIC (MBC) in μg/mL. b Methicillin-resistant strain. NA: not active at maximum concentration tested. NT: not tested.
and A.T.M. acknowledge project funding from the Australian Research Council and AB SCIEX (Concord, Ontario, Canada) through the Linkage Program (LP110200648). References [1] Harden G. Flora of New South Wales, vol. 2. Sydney: UNSW Press; 2002 [revised ed.].
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