PHYTOTHERAPY RESEARCH

Phytother. Res. (2008)

Published online in Wiley InterScience
ANTIBACTERIAL TERPENES FROM THE OLEO-RESIN OF COMMIPHORA MOLMOL (ENGL.) 1
(www.interscience.wiley.com) DOI: 10.1002/ptr.2501

Antibacterial terpenes from the oleo-resin

of Commiphora molmol (Engl.)

M. Mukhlesur Rahman1, Mark Garvey2, Laura J.V. Piddock2 and Simon Gibbons1*
1
Centre for Pharmacognosy and Phytotherapy, the School of Pharmacy, University of London, 29–39 Brunswick Square, London
WC1N 1AX, UK
2
Antimicrobial Agents Research Group, Division of Immunity and Infection, the Medical School, University of Birmingham,
Birmingham, B15 2TT, UK

Two octanordammaranes, mansumbinone (1) and 3,4-seco-mansumbinoic acid (2), and two sesquiterpenes,
β -elemene (3) and T-cadinol (4) have been isolated from the oleo-resin of Commiphora molmol (Engl.). The
structures of these compounds were established unambiguously by a series of 1D and 2D-NMR analyses. We
have also unambiguously assigned all 1H and 13C NMR resonances for 2 and revised its 13C data. The crude
extract of the oleo-resin of C. molmol displayed potentiation of ciprofloxacin and tetracycline against
S. aureus, several Salmonella enterica serovar Typhimurium strains and two K. pneumoniae strains. The
antibacterial activity of terpenes 1–4 was determined against a number of Staphylococcus aureus strains:
SA1199B, ATCC25923, XU212, RN4220 and EMRSA15 and minimum inhibitory concentration (MIC)
values were found to be in the range of 4–256 μg/ml. The highest activity was observed by the seco-A-ring
octanordammarane 2 with an MIC of 4 μg/ml against SA1199B, a multidrug-resistant strain which over-
expresses the NorA efflux transporter, the major characterized antibiotic pump in this species. This activity
compared favorably to the antibiotic norfloxacin with an MIC of 32 μg/ml. Compound 2 also displayed weak
potentiation of ciprofloxacin and tetracycline activity against strains of Salmonella enterica serovar Typhimurium
SL1344 and L10. Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: Commiphora molmol; Burseraceae; octanordammaranes; 3,4-seco-mansumbinoic acid; Antibacterial; Staphylococcus

aureus; MRSA; Salmonella enterica ser. Typhimurium.

or incisions in the bark and is collected as irregular

INTRODUCTION
masses. Myrrh is used as an aromatic for perfumes,
funerals, and insect repellent. It is also used as an anti-
Infections cause by multidrug- and methicillin-resistant septic and anti-inflammatory for the topical treatment
Staphylococcus aureus (MRSA) are cause for some of mouth and throat infections, including gingivitis
concern in the clinical environment. In the UK, MRSA and other gum diseases, tonsillitis and mouth ulcers
has been headline news for the past few years, result- (van Wyk and Wink, 2004). From the phytochemical
ing in considerable public awareness of the potentially perspective, myrrh is known to produce a number of
lethal consequences of an MRSA infection. According sesquiterpenes from its essential oil (Brieskorn and
to the latest data from the Office for National Statistics Noble, 1983; El Ashry et al., 2003; Marongiu et al., 2005;
in England and Wales there has been a dramatic Morteza-Semnani et al., 2001; Zhu et al., 2003).
increase in the number of death certificates that cite Although bacteria can be multi-drug resistant (MDR)
MRSA, with 1211 citations in 2001 rising to 2083 in via multiple mechanisms of resistance, one single mecha-
2005 (Office for National Statistics, 2007). Despite the nism is particularly important i.e. the constitutive over-
release of agents such as linezolid, daptomycin and expression of chromosomally encoded efflux pumps
synercid, the ability of MRSA to acquire resistance (Piddock, 2006). As efflux pumps exist in antibiotic
necessitates further antibacterial discovery. As part susceptible and resistant bacteria they are potential
of a continuing project to characterize new anti- targets for novel non-antibiotic therapeutics. Inhibitors
staphylococcal agents (Smith et al., 2007; Shiu and of various efflux pump systems have been described in
Gibbons, 2006), we have conducted bioassay-guided the literature and typically these are plant alkaloids,
fractionation of the oleo-resin of Commiphora molmol. but as yet no product has been marketed for use in
Commiphora molmol Engl. (syn. Commiphora myrrha human medicine (Lomovskaya and Bostain, 2006).
Holmes, Balsamodendron myrrha; (Burseraceae)) is As a part of an effort to characterize new anti-
a thorny shrub or small tree of approximately 3 m in bacterials with activity against effluxing MDR strains
height that is indigenous to the desert areas of Somalia, of Staphylococcus aureus and Enterobacteriaceae, we
Ethiopia and parts of Kenya (van Wyk and Wink, 2004). report the isolation of four terpenes (1–4) from the oleo-
The oleo-gum resin (oleo-resin) exudes from fissures resin from the stems of Commiphora molmol (Engl.)
as well as their anti-staphylococcal activity against a
panel of drug-resistant Staphylococcus aureus strains.
* Correspondence to: Professor Simon Gibbons, Centre for Pharmacognosy
and Phytotherapy, the School of Pharmacy, University of London, 29–39
We have also unambiguously assigned all 1H and 13C
Brunswick Square London WC1N 1AX, UK. NMR resonances for compound 2 and revised its 13C
E-mail: simon.gibbons@pharmacy.ac.uk data.
Copyright © 2008 John Wiley & Sons, Ltd. Received 12 December
Phytother. 2007
Res. (2008)
Accepted 29DOI:
December 2007
10.1002/ptr
Copyright © 2008 John Wiley & Sons, Ltd.
2 M. M. RAHMAN ET AL.

et al., 2000); SL1344 isolated from calves (Wray and

Sojka, 1986); two laboratory mutants of S. Typhimurium
in which the tolC or acrB genes had been disrupted
(Eaves et al., 2004). In addition, one strain of Staphylo-
coccus aureus (NCTC 8532), two strains of Klebsiella
pneumoniae (NCTC 10896; NCTC 9633), one strain of
Enterobacter cloacae (NCTC 10005), one strain of Serratia
marcescens (NCTC 2847), one strain of Providentia
stuarti (NCTC 10318), one strain of Pseudomonas
aeruginosa (NCTC 10662) and two strains of Escherichia
coli (NCTC 10418 and NCTC 10538) were used. All
strains were grown on Iso-Sensitest medium (Unipath)
MATERIALS AND METHODS aerobically at 37 °C for 18–24 h. All strains were stored
on ProtectTM beads (Technical Service Consultants,
General. NMR spectra were recorded on a Bruker Lancashire, UK) at −80 °C.
AVANCE 500 MHz spectrometer. Chemical shifts
values (δ ) were reported in parts per million (ppm) Minimum inhibitory concentration (MIC) assay. The
relative to the appropriate internal solvent standard MIC of each antibiotic and disinfectant +/– the oleo-
and coupling constants (J values) are given in Hertz. resin of Commiphora molmol or 3,4-seco-mansumbinoic
Vacuum-liquid chromatography (VLC) was carried out acid (2) for each strain of Gram-negative bacteria was
using Merck Si gel 60 H. TLC and preparative-TLC determined by the agar doubling dilution method
were conducted on normal-phase Merck Si gel 60 PF254 according to the guidelines of the BSAC (Andrews,
plates. Spots on TLC and PTLC plates were visualized 2001). All antibiotics and disinfectants were made up
after spraying with 1% vanillin-H2SO4 followed by heat- and used according to the manufacturer’s instructions:
ing at 110 °C for 5–10 min. chloramphenicol, ciprofloxacin, norfloxacin, nalidixic
acid and tetracycline (Sigma-Aldrich Company Ltd,
Plant material. The oleo-resin of C. molmol (Batch Dorset, UK); triclosan (Gift from Ciba, Switzerland).
No. 19214; Origin North Africa) was purchased from The MIC of each antibiotic and disinfectant +/– the
Proline Botanicals, UK, in October 2006. plant extract Commiphora molmol for each strain of
Gram-negative bacteria was also determined by the
Extraction and isolation. 100 g of dried, ground resin of micro-broth dilution following the BSAC recommended
C. molmol was extracted with CHCl3 at room tempera- protocol (Andrews, 2001).
ture. The CHCl3 extract (10.8 g) was fractionated by All 5 S. aureus strains were cultured on nutrient agar
VLC on Silica gel 60H using a mobile phase of petro- (Oxoid) and incubated for 24 h at 37 °C prior to MIC
leum ether, EtOAc and MeOH in order of increasing determination. Norfloxacin was purchased from the
polarity. The eluates were combined together on the Sigma Chemical Co. Mueller-Hinton broth (MHB; Oxoid)
basis of TLC analysis. Preparative TLC (100% hexane; was adjusted to contain 20 and 10 mg/l of Ca2+ and Mg2+,
double development) on the VLC fraction eluted with respectively. An inoculum density of 5 × 105 cfu of each
5% EtOAc in petroleum ether afforded 3 (4.2 mg). VLC of the test organisms was prepared in normal saline
fractions eluted with 10–12.5% EtOAc in petroleum (9 g/l) by comparison with a 0.5 MacFarland standard.
ether were further subjected to preparative-TLC (mobile MHB (125 μl) was dispensed into 10 wells of a 96 well
phase, Toluene:EtOAc:Acetic Acid = 95:4:1, double microtitre plate (Nunc, 0.3 ml volume per well). A stock
development) to yield 1 (8.3 mg) and 4 (7.5 mg). Com- solution of norfloxacin was prepared by dissolving the
pound 2 (10.4 mg) was isolated from the VLC eluted antibiotic in DMSO (Sigma) and dilution in MHB to
with 20–25% EtOAc in petroleum ether followed by give a final concentration of 0.625%. A DMSO control
preparative TLC (mobile phase, Toluene: EtOAc:Acetic was included in all assays.
Acid = 90:9:1, double development). Compounds were serially diluted into each of the wells
followed by the addition of the bacterial inoculum and
Bacterial strains. The antibacterial assay was performed the microtitre plate was incubated at 37 °C for 18 h. The
against a panel of multi-drug and methicillin-resistant MIC recorded the lowest concentration at which no
strains of Staphylococcus aureus. S. aureus standard growth was observed. This was facilitated by the addi-
strain ATCC 25923 and tetracycline-resistant strain tion of 20 μl of a 5 mg/ml methanolic solution of 3-[4,5-
XU212 which possesses the TetK tetracycline efflux dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
protein were provided by Dr Edet Udo (Gibbons and (MTT; Sigma) to each of the wells and incubation for
Udo, 2000). Strain SA-1199B which over-expresses the 20 minutes. A blue colouration indicated bacterial growth.
norA gene encoding the NorA MDR efflux pump was All MICs were determined on at least three inde-
provided by Professor Glenn Kaatz (Kaatz et al., 1993). pendent occasions.
Strain RN4220 which possesses the MsrA macrolide
efflux protein was provided by Dr Jon Cove (Ross
et al., 1989). EMRSA-15 (Richardson and Reith, 1993)
was the generous gift of Dr Paul Stapleton. RESULTS AND DISCUSSION
In addition, ten strains of Salmonella enterica serovar
Typhimurium strains were used (Table 2). The strains Chemistry
consisted of S. Typhimurium L3, which was isolated
from a patient prior to a course of ciprofloxacin, four VLC fractionation of a chloroform extract of the oleo-
post-therapy strains (L10–L18) were MDR (Piddock resin from the stems of Commiphora molmol (Engl.)
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. (2008)
DOI: 10.1002/ptr
 ANTIBACTERIAL TERPENES FROM THE OLEO-RESIN OF COMMIPHORA MOLMOL (ENGL.) 3

Table 1. 1H NMR (500 MHz), 13C NMR (125 MHz) and HMBC (500 MHz) data of 2 in CDCl3

HMBC (H→C)
1 13 2 2
Position H C J J

1 1.63, m 34.5 – –
2 2.42, m 28.4 – –
3 – 179.9 – –
4 – 147.7 – –
5 2.00, dd, J = 12.4, 2.7 Hz 51.2 C-4 C-7, C-19, C-20, C-22
6 1.38, m 25.0 – –
7 1.65, m 34.4 – –
8 – 39.8 – –
9 1.88, m 41.7 – C-5, C-12, C-19
10 – 39.5 – –
11 1.73, m 22.6 – –
12 1.46, m 24.0 – –
13 2.77, br d, J = 12.4 Hz 47.9 – C-16
14 – 53.6 – –
15 2.38, m 40.2 – –
16 5.67, m 130.2 C-15, C-17 C-13, C-14
17 5.60, m 134.2 C-13, C-16 C-14, C-15
18 1.08, s 17.2 – C-7, C-9, C-14
19 0.88, s 20.2 C-10 C-1, C-5, C-9
20 1.75, s 23.4 C-4 C-5, C-22
21 1.03, s 18.1 C-14 C-13, C-8
22 4.69, d, J = 1.5 Hz 113.7 C-4 C-5, C-20
4.89, d, J = 1.5 Hz

yielded two octanordammaranes and two sesquiterpenes. of the HMBC spectra which revealed 3J connectivities
By comparing the spectral data to those previously by both H-5 and H-9 to a methyl carbon at 20.2
reported, the compounds were identified as β-elemene (Me-19) and another 3J correlation by H-5 and the exo-
(3) (Adio et al., 2004), T-cadinol (4) (Claeson et al., methylene protons to a carbon at 23.4 (Me-20). The
1991) and mansumbinone (1) (Provan and Waterman, assignments of C-16 and C-17 at 129.8 and 133.9 were
1986). The remaining octanordammarane was identi- also reported to be interchangeable by Provan and
fied as 3,4-seco-mansumbinoic acid (2). Although the Waterman (1986). We were able to resolve this ambi-
1
H and 13C NMR data of 2 was reported previously by guity by careful analysis of the HMBC (Table 1) and
Provan and Waterman (1986), there was ambiguity in COSY spectra. A 3J correlation between the hydrogens
the assignment of several carbon chemical shifts. A series of methyl-21 and C-13 enabled assignment of H-13 (δH
of 1D and 2D NMR data including COSY, NOESY, = 2.77, br d) via the HMQC spectrum. H-13 exhibited
HMQC and HMBC has enabled us to revise its 13C a COSY correlation to H-17 (δH 5.60) and its associ-
NMR data here. ated carbon resonance could be discerned by analysis
The 1H NMR (500 MHz, CDCl3, Table 1) of 2 showed of the HMQC spectrum (C-17; δC 134.2). H-17 was also
the presence of an exo-methylene (4.69, d, J = 1.5 Hz; coupled to H-16 (δH 5.67) and we were again able to
4.89, d, J = 1.5 Hz), two olefinic hydrogens (5.60, m; identify C-16 on the basis of direct correlation observed
5.67, m), four methyl groups (0.88, s; 1.03, s; 1.08, s; in the HMQC spectrum (δC 130.2). We were therefore
1.75, s) and a group of unresolved methine and able to unambiguously revise the assignment of carbons
methylene hydrogens. The 13C NMR (125 MHz, CDCl3, C-16 and C-17 to 130.2 and 134.2, respectively. The
Table 1) of 2 exhibited a total of 22 carbons including chemical shifts of the remaining carbon resonances of 2
a carboxylic acid (δC 179.9). The DEPT135 experiment were almost identical to those described by Provan and
revealed the presence of 8 methylene carbons includ- Waterman (1986). Therefore, compound 2 was identi-
ing an exo-methylene at 113.7 ppm. In the HMBC fied as 3,4-seco-mansumbinoic acid previously isolated
spectrum (Table 1), a common 3J correlation by two from Commiphora incisa. Among these four com-
methyl groups resonating at 0.88 and 1.75 and the exo- pounds, this is the first time report of isolation of 1 and
methylene protons to a methine carbon at 51.2 ppm 2 from C. molmol.
confirmed its assignment as C-5. Again, C-9 was
assigned at 41.7 ppm by 3J correlations from the methyl
hydrogens at 1.08 (Me-18) and 0.88 (Me-19). The Anti-staphylococcal activity
carbon chemical shifts of C-5 and C-9 were previously
reported (Provan and Waterman, 1986) at 41.4 and 51.0, All compounds were assessed for their in vitro anti-
respectively, which we have revised here. staphylococcal activity in a MIC assay (Table 2). Among
Provan and Waterman (1986) assigned the carbon the sesquiterpenes and octanordammaranes, the most
chemical shifts of Me-19 and Me-20 at 19.9 and 23.1 pronounced anti-staphylococcal activity was exhibited
and reported these to be interchangeable. However, by compound 2. Its highest potency was observed
we can confirm the assignment of Me-19 and Me-20 at against the multi-drug-effluxing strain SA 1199B (MIC
20.2 and 23.4, respectively. This was achieved by analysis 4 μg/ml) and was found to be eight times more potent
Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. (2008)
DOI: 10.1002/ptr
 4

Table 2. MICs of antibiotics and terpenes +/– CM to S. Typhimurium, K. pneumoniae and S. aureus.

Copyright © 2008 John Wiley & Sons, Ltd.

MIC μg/ml−1
cip nor tet
Strain Species Description Reference cip (+CM) nor (+CM) tet (+CM) 1 2 3 4

L354 S. Typhimurium SL1344, commonly used UK strain Wray & Sojka, 1978 0.03 0.03 0.25 0.25 2 2 nd 0.015 nd nd
14028s S. Typhimurium Commonly used wild-type strain Type strain 0.03 0.03 0.12 0.12 4 2 nd nd nd nd
LT2 S. Typhimurium Commonly used wild-type strain Type strain 0.03 0.03 0.12 0.12 4 2 nd nd nd nd
L3 S. Typhimurium Human pre-therapy clinical isolate, susceptible Piddock et al., (2000) 0.03 0.015 0.06 0.06 1 1 nd nd nd nd
L10 S. Typhimurium Human post-therapy clinical isolate, acrAB+++ Piddock et al., (2000) 0.25 0.12 0.5 0.5 16 8 nd 0.12 nd nd
L12 S. Typhimurium Human post-therapy clinical isolate Piddock et al., (2000) 0.5 0.5 2 2 8 4 nd nd nd nd
L13 S. Typhimurium Human post-therapy clinical isolate Piddock et al., (2000) 0.5 0.25 2 2 16 8 nd nd nd nd
L18 S. Typhimurium Human post-therapy clinical isolate, acrAB+++ Piddock et al., (2000) 0.5 0.5 2 2 32 16 nd nd nd nd
L108 S. Typhimurium L354 tolC::aph Buckley et al., (2006) 0.008 0.008 0.06 0.03 1 1 nd nd nd nd
L643 S. Typhimurium L354 acrB::aph Eaves et al., (2004) 0.008 0.008 0.03 0.03 2 1 nd nd nd nd
F77 S. aureus NCTC 8532 type strain Type strain 0.25 0.12 1 0.5 4 2 nd nd nd nd
ATCC 25923 S. aureus Commonly used wild-type strain Type strain nd nd 1 nd nd nd 128 64 256 256
M. M. RAHMAN ET AL.

XU212 S. aureus TetK tetracycline efflux protein Gibbons and Udo, 2000 nd nd 16 nd nd nd 64 32 128 128
SA-1199B S. aureus NorA MDR efflux pump Kaatz et al., 1993 nd nd 32 nd nd nd 64 4 128 256
RN4220 S. aureus MsrA macrolide efflux protein Ross et al., 1989 nd nd 2 nd nd nd 256 16 64 64
EMRSA-15 S. aureus Epidemic methicillin-resistant strain Richardson and Reith, 1993 nd nd 1 nd nd nd 128 16 128 64
H42 K. pneumoniae NCTC 10896 type strain Type strain 0.12 0.06 2 0.5 8 4 nd nd nd nd
H43 K. pneumoniae NCTC 9633 type strain Type strain 0.25 0.12 2 1 8 4 nd nd nd nd

Bold represents a reduction in the MIC of the antibiotic on the addition of Commiphora molmol. Cip, ciprofloxacin; nor, norfloxacin; tet, tetracycline. (+CM) = 200 μg/ml of Commiphora molmol
added. acrAB+++, over-expression of the genes acrAB. nd, not determined.

DOI: 10.1002/ptr
Phytother. Res. (2008)
 ANTIBACTERIAL TERPENES FROM THE OLEO-RESIN OF COMMIPHORA MOLMOL (ENGL.) 5

than the control antibiotic norfloxacin. It is possible was observed on the MIC of norfloxacin on any of the
that this metabolite is a fatty acid mimic, possessing a other strains of Enterobacteriaceae tested (data not
carboxylic acid function due to the formation of the shown).
seco-A-ring moiety and 2 may interfere with biosynthesis The oleo-resin of C. molmol (200 μg ml−1) had a
of essential fatty acids. broader affect with tetracycline compared to the other
agents tested (Table 2). The MIC of tetracycline was
reduced by 2-fold in the presence of the oleo-resin
Antibacterial activity for Gram-negative bacteria (200 μg ml−1) for the wild-type S. Typhimurium strains
14028s and LT2, the post therapy human isolates, L10,
The wild-type strains of S. Typhimurium (L354, 14028s L12, L13 and L18, L643 with acrB disrupted, the S.
and LT2) showed the typical susceptibility of this aureus strain F77 and the two K. pneumoniae strains
species to all agents tested (Table 2). H42 and H43 (Table 2). As previously observed
The oleo-resin of C. molmol (200 μg ml−1) reduced for norfloxacin, no affect was observed on the MIC
the MIC of ciprofloxacin by 2-fold for the human of tetracycline on any of the other strains of Enter-
pre-therapy isolate (L3), two of the post-therapy MDR obacteriaceae tested (data not shown).
strains (L10 and L13), S. aureus NCTC 8532 and Out of the four compounds isolated from the oleo-
K. pneumoniae NCTC 10896 and 9633 (Table 2). The resin of C. molmol only compound 2 displayed a
oleo-resin of C. molmol had no affect on the MIC of weak 2-fold potentiation of ciprofloxacin and tetracyc-
ciprofloxacin on any of the other strains of Entero- line against S. Typhimurium strains SL1344 and L10
bacteriaceae tested (data not shown). (Table 2). No other Enterobacteriaceae were affected
The oleo-resin of C. molmol (200 μg ml−1) reduced (data not shown).
the MIC of norfloxacin by 2-fold for the two laboratory
mutants with the tolC (L108) or acrB (L643) genes
disrupted; however it had no effect on any other of the Acknowledgement
S. Typhimurium strains. The oleo-resin reduced the MIC
of norfloxacin by four 2-fold for S. aureus and the two We thank the Leverhulme Trust for the award of postdoctoral
K. pneumoniae strains (Table 2). However, no affect fellowships to M. M. Rahman and M. Garvey (Grant No. F/00094AR).

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Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. (2008)
DOI: 10.1002/ptr