Fitoterapia 105 (2015) 269–272

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Fitoterapia

journal homepage: www.elsevier.com/locate/fitote

Antibacterial constituents of Neohyptis paniculata

M. Mukhlesur Rahman a,b,⁎, Simon Gibbons a
a
Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, 29–39 Brunswick Square, London WC1N 1AX, UK
b
Medicine Research Group, School of Health, Sport and Bioscience, University of East London, Stratford Campus, Water Lane, London E15 4LZ, UK

a r t i c l e i n f o a b s t r a c t

Article history: A new α-pyrone, 6R-[5R,6S-diacetyloxy-1Z,3E-heptadienyl]-5,6-dihydro-2H-pyran-2one (1), along with six

Received 13 November 2012 known compounds including an α-pyrone, flavones and terpenes was isolated from the aerial parts of Neohyptis
Received in revised form 8 July 2015 paniculata. The structure of 1 was established unambiguously by MS and a series of 1D and 2D-NMR spectroscop-
Accepted 9 July 2015
ic analyses. The antibacterial activity of the compounds (1–7) was investigated against five strains of multi-drug
Available online 21 July 2015
resistant (MDR) and methicillin-resistant Staphylococcus aureus and minimum inhibitory concentrations (MICs)
Keywords:
of these compounds were found to be in the range of 64–256 μg/mL.
Neohyptis paniculata © 2015 Elsevier B.V. All rights reserved.
Lamiaceae
α-Pyrone
6R-[5R,6S-diacetyloxy-1Z,3E-heptadienyl]-5,6-
dihydro-2H-pyran-2one
Antibacterial
Staphylococcus aureus

1. Introduction 2. Materials and methods

Neohyptis paniculata (Bak.) J. K. (Lamiaceae), a slender and erect pe- 2.1. General
rennial herb attaining a height of 1–3 ft., is a monotypic species that is
characterized by a small and white corolla with purple dots. The plant Optical rotations were measured on a Perkin Elmer Polarimeter
grows well in savanna swamps of some African countries including 341. IR spectra were recorded as a dry film on a Perkin Elmer
Ghana, Guinea, Angola, Cameroon, Angola and Sierra-Leone [1]. This Spectrum 1000 FT-IR spectrometer. UV spectra were obtained on a
species has not been previously investigated for phytochemistry and Unicam UV 4–100 UV/vi spectrophotometer in MeOH. HREIMS was
bioactivity. recorded on a Micromass Q-TOF Global Tandem Mass Spectrometer.
Infections caused by multidrug-resistant (MDR) and methicillin- NMR spectra (both 1D and 2D) were obtained on a Bruker AVANCE
resistant strains of Staphylococcus aureus (MRSA) are still problematic 500 spectrometer (500 MHz for 1H and 125 MHz for 13C), using the
in the clinical environment and the need for new antibacterials is residual solvent peaks as internal standard. Vacuum-liquid chroma-
becoming increasingly urgent. As part of an on-going effort to character- tography (VLC) was carried out using Merck Si gel 60 H. Gel filtration
ize new compounds from species of Lamiaceae [2,3] with antibacterial was performed using Sephadex LH-20 (Sigma). TLC and PTLC were
activity against multidrug-resistant (MDR) strains of S. aureus, we here conducted on normal-phase Merck Si gel 60 PF 254 and reverse-
report the isolation of a new compound (1) together with six known phase Merck Si gel RP-18 PF 254 plates (20 cm × 20 cm). Spots on
compounds including an α-pyrone, flavones and terpenes from the TLC and PTLC plates were visualised under UV light (254 and
aerial parts of N. paniculata and the antibacterial activities of compounds 366 nm) and spraying with 1% vanillin-H2SO4 followed by heating
1–7 against five bacterial strains of MDR and methicillin-resistant at 110 °C for 5–10 min.
S. aureus.

2.2. Plant material

⁎ Corresponding author at: Medicine Research Group, School of Health, Sport and
The aerial parts of N. paniculata were collected from Ghana in 2006.
Bioscience, University of East London, Stratford Campus, Water Lane, London E15 4LZ, UK. A herbarium specimen of this collection is maintained at the UCL School
E-mail address: m.rahman@uel.ac.uk (M.M. Rahman). of Pharmacy, London, UK.

http://dx.doi.org/10.1016/j.fitote.2015.07.012
0367-326X/© 2015 Elsevier B.V. All rights reserved.
270 M.M. Rahman, S. Gibbons / Fitoterapia 105 (2015) 269–272

2.3. Extraction and isolation 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT;

Lancaster) was used to detect bacterial growth by a colour change
250 g of dried, ground plant material was sequentially extracted from yellow to blue [8].
with hexane, CHCl3 and methanol in a Soxhlet apparatus. This sequen-
tial extraction with solvents of increasing polarity allowed preliminary 3. Results and discussion
separation of the components based on the polarity of the metabolites.
As both hexane and chloroform extracts showed antibacterial activity, The HREIMS of 1 showed a [M + Na] peak at m/z 331.1157 (calc.
these two extracts were analysed further for the isolation and purifica- 331.1163) and thereby, established its molecular formula as C16H20O6.
tion of compounds. The 13C and DEPT135 NMR experiments revealed the presence of 16
The hexane extract (900 mg) was subjected to gel filtration over carbons; three methyls (15.2, 21.3 and 21.4), one methylene (29.9),
Sephadex LH20 (27 g) using a mobile phase of hexane, CHCl3 and three carbonyls (164.0, 170.3 and 170.6) and nine methines, three of
MeOH in a ratio of 2:5:1 to obtain a total of 8 fractions (50 mL each frac- which were oxygenated. The 1H NMR spectrum (500 MHz, CDCl3,
tion). Solid phase extraction (SPE) (Si 60G; mobile phase 100% hexane Table 1) of 1 showed two acetoxy methyl groups which resonated at
to 100% EtOAc) on fraction 2 (200 mg) was carried out using hexane 2.06 (3H, s) and 2.09 (3H, s) and a further 3H signal at 1.22 (3H, d,
and EtOAc in a mixture of increasing polarity collecting 50 mL of each J = 6.5 Hz) which was attributable to a terminal secondary methyl
eluent. Preparative TLC (Si gel 60 PF254; 100% hexane) on the SPE sub- group. The spectrum also exhibited two olefinic protons of a
fraction eluted with 100% hexane afforded 2 (2.2 mg) and 3 (3.1 mg) dihydropyrone skeleton [9] at 6.09 (dq, J = 10.0, 1.0 Hz) and 6.92
while compounds 1 (2.5 mg) and 4 (10.6 mg) were isolated from (ddd, J = 10.0, 5.5, 3.0 Hz), two cis olefinic protons (5.64, dd, J = 10.5,
the SPE sub-fraction eluted with 50% EtOAc in hexane followed by 9.0 Hz; 6.18, dd, J = 11.5, 10.5 Hz) and two trans olefinic protons
preparative-TLC (Merck Si gel RP-18 PF254; MeOH: H2O: AcOH = (6.52, dd, J = 15.0, 11.5 Hz; 5.77, dd, J = 15.0, 7.0 Hz), one methylene
59:40:1). Further, preparative TLC (Si gel 60 PF254; 15% EtOAc in toluene (2.44, m) and three oxy-methines (5.08, 5.37 and 5.45). The unambigu-
plus 3–4 drops of AcOH) on the SPE sub-fraction eluted with 10–20% ous assignment of all carbons and protons of 1 was achieved by a series
EtOAc hexane led to the isolation of 5 (4.1 mg) and 7 (6.3 mg). of 2D experiments including HMQC, HMBC, COSY and NOESY. In the
The CHCl3 extract (5.5 g) was fractionated by VLC over Si gel 60H HMBC experiment, the proton at 6.92 (H-4) showed 3J connectivity to
using hexane–EtOAc and EtOAc–MeOH mixtures of increasing polarity. a carbonyl at 164.0 (C-2), and to an oxygenated methine carbon at
The eluates were combined together on the basis of TLC analysis. VLC 74.0 (C-6; δH 5.37 from HMQC). In the COSY experiment, H-4 revealed
fractions eluted with 20–25% EtOAc in petroleum ether were further an expected interaction with H-3 (δH 6.09; δC 121.9) and H2-5 (2.44;
subjected to preparative-TLC to yield 5 (14.5 mg) and 6 (16.6 mg). Gel δC 29.9 from HMQC). These chemical shifts and their correlations sup-
filtration over Sephadex LH20 (hexane: CHCl3: MeOH = 2:5:1) on the ported the presence of 5,6-dihydro-2H-pyran-2-one as a part of the
VLC fractions eluting with 40–50% EtOAc in hexane gave 15.4 mg of 7. molecule. The olefinic proton at 5.64 (H-1′; δC 128.8 from HMQC)
coupled to H-6 and H-2′ (δH 6.18; δC 131.4) in the COSY experiment
2.4. 6R-[5R,6S-diacetyloxy-1Z,3E-heptadienyl]-5,6-dihydro-2H-pyran- and showed 3J HMBC connectivity to a methylene carbon at 29.9 (C-5)
2one (1) and to an olefinic carbon at 128.4 (C-3′; δH 6.52). The latter proton
(H-3′) coupled to H-2′ and H-4′ (δH 5.77; δC 130.6) in the COSY exper-
White gum. [α]20 MeOH
D − 30.1 (CHCl3; c 4.55). UV λ max nm (logε): 229 iment and exhibited a 3J HMBC correlation to C-1′ and to an oxygenated
−1
(4.26). IR (solution in CHCl3) ν cm : 1736, 1360, 1225, 1070, 1025, methine 74.8 (C-5′). The remaining oxy-methine proton at 5.08 (H-6′)
955, 800; 1 H NMR, 13 C NMR and HMBC, see Table 2. HRMS m/z revealed a COSY interaction with H-5′ and to a methyl doublet (J =
[M + Na] peak at m/z 331.1157 (Calcd. 331.1163). 6.5 Hz) at 1.22 (H3-7′; δC15.2 from HMQC). The remaining methyls at
2.06 and 2.09 ppm showed 2J HMBC connectivity to their respective
2.5. Bacterial strains acetyl carbonyl carbons at 170.3 and 170.6. The 3J HMBC correlation
by H-5′ and H-6′ to the carbonyls at 170.6 and 170.3 confirmed their
A standard S. aureus strain ATCC 25923 and a clinical isolate connectivity via C-5′ and C-6′, respectively. Accordingly, compound 1
(XU212), which possesses the TetK efflux pump and is also an MRSA was therefore identified as 6ζ-[5ζ,6ζ-diacetyloxy-1Z,3E-heptadienyl]-
strain, were obtained from E. Udo [4]. Strain RN4220 which has the 5,6-dihydro-2H-pyran-2one (1, Fig. 1; named neohyptolide) which is
MsrA macrolide efflux pump was provided by J. Cove [5]. EMRSA-15 a new compound. Because of paucity of the compound, it was not pos-
[6] was obtained from Dr Paul Stapleton, UCL. Strain SA1199B which sible to carry out further study to confirm the absolute stereochemistry
over-expresses the NorA MDR efflux pump was the gift of Professor
Glenn Kaatz [7]. Norfloxacin was obtained from the Sigma Chemical Co.
Table 1
1
H NMR (500 MHz), 13C NMR (125 MHz) and HMBC data of 1 in CDCl3.
2.6. Antibacterial assay
1 13
Position H C HMBC

Overnight cultures of each strain were made up in 0.9% saline to an 2

J 3
J
inoculum density of 5 × 105 cfu/mL by comparison with a MacFarland 2 – 164.0 – –
standard. Tetracycline and oxacillin were dissolved directly in MHB, 3 6.09, dq, J = 10.0, 1.0 Hz 121.9 C–2, C–4 C–5
whereas norfloxacin and erythromycin were dissolved in DMSO and 4 6.92, ddd, J = 10.0, 5.5, 3.0 Hz 144.8 C–3 C–2, C–6
then diluted in MHB to give a starting concentration of 512 μg/mL. 5 2.44, m 29.9 C–4 C–3, C–1′
6 5.37, ddt, J = 10.0, 5.0, 1.0 Hz 74.0 – C–4, C–2′
Using Nunc 96-well microtitre plates, 125 μL of MHB was dispensed 1′ 5.64, dd, J = 10.5, 9.0 Hz 128.8 C–2′ C–5, C–3′
into wells 1–11. 125 μL of the test compound or the appropriate antibi- 2′ 6.18, dd, J = 11.5, 10.5 Hz 131.4 C–1′, C–3′ C–6, C–4′
otic was dispensed into well 1 and serially diluted across the plate 3′ 6.52, dd, J = 15.0, 11.5 Hz 128.4 C–2′, C–4′ C–1′, C–5′
leaving well 11 empty for the growth control. The final volume was 4′ 5.77, dd, J = 15.0, 7.0 Hz 130.6 C–3′, C−5′ C–4′, C−6′
5′ 5.45, ddd,, J = 7.0, 3.5, 1.0 Hz 74.8 C–6′ C–3′, C−7′
dispensed into well 12, which being free of MHB or inoculum served
6′ 5.08, dq, J = 6.5, 3.5 Hz 70.7 C–5′ C–4′
as the sterile control. Finally, the bacterial inoculum (125 μL) was 7′ 1.22, d, J = 6.5 Hz 15.2 C–6′ C–5′
added to wells 1–11 and the plate was incubated at 37 °C for 18 h. A CH3CO-5′ 2.09, s 21.3 CO–5′ –
DMSO control (3.125%) was also included. All MICs were determined CH3CO-6′ 2.06, s 21.4 CO–6′ –
in duplicate. The MIC was determined as the lowest concentration at CH3CO-5′ – 170.6 – –
CH3CO-6′ – 170.3 – –
which no growth was observed. A methanolic solution (5 mg/mL) of
 M.M. Rahman, S. Gibbons / Fitoterapia 105 (2015) 269–272 271

1' 5
2' 6
4

3'
O 3
H
1 2
4'

O 5'
O
6' O
O H
7'
O
1 2

H
O
H

O O
O
O
O
3 4

OMe
H
MeO O
O
H OR
OH O
5 6; R=H
7; R=Me
Fig. 1. Structures of compounds 1–7.

of the compound. However, R-configuration was proposed at C-5 by All compounds (1–7) were assessed for anti-staphylococcal activity
comparing its 1H NMR data to other structurally related hyptolides [9, in a minimum inhibitory concentration (MIC) assay but displayed only
10]. Again, in view of the similarity of the coupling constants of the side weak to moderate activity (Table 2). Among the compounds, the com-
chain protons at C-5′ and C-6′ of 1 to hyptolides reported from Hyptis paratively better anti-staphylococcal activity was exhibited by compound
oblongifolia [9] and pectinolides from Hyptis pectinata [11], the configura- 1. α-Pyrones with structural similarity to 1 have been reported to exhibit
tions of these chiral centres of 1 were assumed to be the same as in those antibacterial properties including those against the clinical isolates of
compounds, i.e., R-configuration at C-5′ and S-configuration at C-6′. So Staphylococcal aureus [16]. The cytotoxic potential of pectinolides was
taking these configurations into account, compound 1 was considered also reported with a number of cultured cell lines [16]. It is suggested
to be 6R-[5R,6S-diacetyloxy-1Z,3E-heptadienyl]-5,6-dihydro-2H-pyran- that the presence of an alpha-beta unsaturated ketone, which is part of
2one. the lactone moiety may make this compound a substrate for biological
By direct comparison of spectral data to those published in the nucleophiles and it is likely that this compound is also cytotoxic to mam-
literature, compounds 2–7 were identified as α-himachalene (2) [12], malian cells as is seen in the case of the pectinolides [16].
4-deacetoxy-10-epi-olguine (3) [9], isoneocembrene-A (4) [13], β-
caryophyllene oxide (5) [14], 3,5-dihydroxy-7,4′-dimethoxyflavone
Conflict of interest
(6) [15], and 5-hydroxy-3,7,4′-trimethoxyflavone (7) [15].
There is no conflict of interest.
Table 2
Minimum inhibitory concentrations (μg/mL) of compounds 1–7 against clinical isolates of
Acknowledgement
multi-drug resistant (MDR) and methicillin-resistant strains of Staphylococcus aureus.

Compounds SA1199B RN4220 EMRSA-15 XU-212 ATCC25923 MMR is grateful to the Leverhulme Trust for the award of postdoc-
1 64 64 64 64 128 toral research fellowship (DD-LF).
2 64 64 128 64 128
3 64 128 64 128 128
4 64 128 128 64 128 References
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