Journal of Ethnopharmacology 126 (2009) 500–505

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Antimycobacterial terpenoids from Juniperus communis L. (Cuppressaceae)

Andréa Y. Gordien a , Alexander I. Gray a , Scott G. Franzblau b , Véronique Seidel a,∗
a
Natural Products Research Laboratories, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, UK
b
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA

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

Article history: Ethnopharmacological relevance: Juniperus communis is a plant which has been reported as a traditional
Received 24 June 2009 cure for tuberculosis (TB) and other respiratory diseases.
Received in revised form 26 August 2009 Aim of the study: The aim of this study was to isolate and identify the constituents responsible for the
Accepted 5 September 2009
activity of the n-hexane extract of Juniperus communis roots against Mycobacterium tuberculosis H37 Rv
Available online 13 September 2009
and Juniperus communis aerial parts against Mycobacterium aurum. Subsequently, it was to evaluate the
activity of the pure isolated compounds against (i) drug-resistant Mycobacterium tuberculosis variants,
Keywords:
(ii) non-replicating Mycobacterium tuberculosis and (iii) a range of non-tuberculous mycobacteria (NTM).
Antimycobacterial activity
Juniperus communis
Materials and methods: The antimycobacterial activity of Juniperus communis extracts, fractions and con-
Mycobacterium tuberculosis stituents was determined against Mycobacterium tuberculosis H37 Rv, and against rifampicin-, isoniazid-,
Longifolene streptomycin- and moxifloxacin-resistant variants, using the microplate broth Alamar Blue assay (MABA)
Totarol method. Isolated constituents were tested against non-replicating Mycobacterium tuberculosis H37 Rv,
Trans-communic acid using the low oxygen recovery assay (LORA), and against NTM (Mycobacterium aurum, Mycobacterium
phlei, Mycobacterium fortuitum and Mycobacterium smegmatis), using a broth microdilution method.
Cytotoxicty studies were performed using mammalian Vero cells.
Results: The antimycobacterial activity of Juniperus communis was attributed to a sesquiterpene identi-
fied as longifolene (1) and two diterpenes, characterised as totarol (2) and trans-communic acid (3). All
compounds were identified following analysis of their spectroscopic data (1D- and 2D-NMR, MS) and
by comparison with the literature and commercial authentic standards when available. Revised assign-
ments for 3 are reported. Totarol showed the best activity against Mycobacterium tuberculosis H37 Rv (MIC
of 73.7 ␮M). It was also most active against the isoniazid-, streptomycin-, and moxifloxacin-resistant
variants (MIC of 38.4, 83.4 and 60 ␮M, respectively). Longifolene and totarol were most active against
the rifampicin-resistant variant (MICs of 24 and 20.2 ␮M, respectively). Totarol showed the best activity
in the LORA assay (MIC of 81.3 ␮M) and against all NTM species (MICs in the range of 7–14 ␮M). Trans-
communic acid showed good activity against Mycobacterium aurum (MIC of 13.2 ␮M). The low selectivity
indices (SI) obtained following cytotoxicity studies indicated that the isolated terpenoids were relatively
toxic towards mammalian cells. This is the first report of the isolation of (1) and (2) from Juniperus com-
munis roots, and of (3) from the aerial parts. The antimycobacterial activity of (1) and (3), and the activity
of (2) against Mycobacterium aurum, Mycobacterium fortuitum and Mycobacterium phlei, is reported for the
first time. The effect of totarol on drug-resistant variants and non-replicating Mycobacterium tuberculosis
has never been published.
Conclusions: The presence of antimycobacterial terpenoids in Juniperus communis aerial parts and roots
justifies, to some extent, the ethnomedicinal use of this species as a traditional anti-TB remedy.
© 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction resurgence, accelerated by the HIV/AIDS pandemic, is now posing

With nearly two million deaths annually, tuberculosis (TB) is
currently the leading infectious killer worldwide and it is estimated
that Mycobacterium tuberculosis, the causative agent of TB, has
infected one-third of the world’s population (WHO, 2008). The TB
∗ Corresponding author. Tel.: +44 0141 548 2751; fax: +44 0141 552 2562.
E-mail address: veronique.seidel@strath.ac.uk (V. Seidel).
a serious threat to healthcare systems, especially with the emer-
gence of multidrug- (MDR) and extensively drug-resistant (XDR) TB
(Chan and Iseman, 2008; Gandhi et al., 2006). Current anti-TB treat-
ments involve a long course of a combination of antibiotics with
toxic side effects, and lead to poor patient compliance. This con-
tributes to sustaining drug resistance. There is now an urgent need
to discover and develop new anti-TB drugs particularly to target
drug resistance, and improve the treatment of latent TB by target-
ing tubercle bacilli which are thought to remain within the lungs

0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2009.09.007
 A.Y. Gordien et al. / Journal of Ethnopharmacology 126 (2009) 500–505
in a non-replicating state of persistence (O’Brien and Nunn, 2001).
Additionally, new antimycobacterial agents are needed to improve
the treatment of opportunistic diseases caused by non-tuberculous
mycobacteria (NTM) which are also on the increase (Gillespie et al.,
2001; Saiman, 2004).
Mycobacteria thrive in soils which are wet, acidic and covered
with dense vegetation primarily composed of mosses, ericaceous
plants and conifers (Kazda, 2000). Such an environment is typ-
ical of the boglands of Scotland where many ericaceous plants
and conifers are common. Given the tendency of such plants to
develop in this unique ecosystem despite the mycobacterial chal-
lenge in the soil, we anticipated that they were likely to produce
some antimycobacterial metabolites. In a preliminary screening
of selected Scottish plants for antimycobacterial activity, it was
observed that the n-hexane extract of Juniperus communis roots
showed potent growth inhibition of Mycobacterium tuberculosis
(92.9% at 100 ␮g/mL) whilst the n-hexane extract of Juniperus com-
munis aerial parts was active against Mycobacterium aurum (MIC of
64 ␮g/mL) (Gordien et al., unpublished).
Juniperus communis subsp. communis var. communis L. (Cup-
pressaceae), or common juniper, is a coniferous shrub distributed
throughout the Artic and temperate zone of the Northern hemi-
sphere (Allen and Hatfield, 2004). Its dried blueish-black cones,
known as “juniper berries”, are said to stimulate the appetite and
are used as a flavouring agent for culinary purposes and in the
preparation of gin spirits (Darwin, 2000; Foster, 1999). They have
also been used for various medicinal purposes, including as an
abortifacient, antiseptic, contraceptive, diuretic, and as a remedy
for urinary tract infections, scrofula, chest complaints, diabetes,
rheumatism and backache. The smoke from burnt juniper branches
has been used as a fumigant to prevent the spread of infections
(Allen and Hatfield, 2004; Darwin, 2000; Newton et al., 2002;
Tilford, 1997). More interestingly, juniper has been reported as
a traditional cure for chest troubles such as bronchitis (Kirtikar
and Basu, 2004) and for tuberculosis (McCutcheon et al., 1997; UK
CropNet MPNADB, 2009). The present paper describes the isola-
tion and characterisation of the constituents responsible for the
antimycobacterial activity of common juniper.
2. Materials and methods
2.1. Analytical procedures
Optical rotations were measured using an Autopol V polarimeter
(Rudolph Research Analytical) using a 10 mm cell. IR spectra (film
or KBr) were recorded on a Mattson ATI Genesis FT-IR spectrome-
ter. UV spectra were run on a UNICAM UV 300 spectrophotometer.
High resolution MS data were recorded using either electron impact
(EI) at 70 eV, chemical ionisation (CI) or fast atom bombardment
(FAB) ionisation (using either glycerol or 3-nitrobenzyl alcohol as
a matrix) on a JEOL JMS-700 dual sector mass spectrometer. MS
data using electrospray ionisation (ESI) were recorded on a Finni-
gan LTQ-Orbitrap spectrometer. Both 1 H (400 MHz) and 13 C NMR
(100 MHz) spectra were recorded, in either C5 D5 N or CDCl3 , on
a JEOL Lambda Delta 400 spectrometer. All spectra were refer-
enced on the residual solvent peaks and processed using Mestre
Nova® (MNova) software version 5.3.0 (Mestrelab Research SL,
Spain).
(+)-Totarol (≥97% purity; Sigma–Aldrich, UK) and (+)-
longifolene (98% purity; MP Biomedicals, UK) were used as
authentic standards. TLC analysis of the compounds was per-
formed using silica gel PF254 pre-coated plates (VWR International,
UK). Compounds were detected under short ( = 254 nm) and
long ( = 366 nm) UV light and then visualised by spraying the
plates with p-anisaldehyde sulphuric acid reagent (solution of 0.5%
anisaldehyde–10% glacial acetic acid–5% H2 SO4 in MeOH) followed
501
by heating at 110 ◦ C for 2 min. Vacuum Liquid Chromatography
(VLC) was performed using silica gel 60H (VWR International, UK).
Gel filtration (size exclusion chromatography) was performed
using Sephadex® LH-20-100 (Sigma–Aldrich, UK). All samples
were concentrated to dryness in vacuo at 40 ◦ C and stored at
−20 ◦ C prior to testing.
2.2. Plant material
Juniperus communis L. was purchased from Alba Trees Plc nurs-
ery (Gladsmuir, Scotland) in December 2005. A voucher specimen
(#00140/03) is kept in the Herbarium of the Natural Prod-
uct Research Laboratories, Strathclyde Institute of Pharmacy and
Biomedical Sciences.
2.3. Extraction and isolation
The dried powdered roots of Juniperus communis (217 g) were
subjected to pressurised liquid extraction. The material was mixed
with some sand (50–70 mesh) (Sigma–Aldrich, UK) in a 4:1 ratio,
and the mixture was placed into a sample cell (100 mL). The lat-
ter was loaded into an Accelerated Solvent Extractor ASE 100®
system (Dionex, UK). Extractions were performed under pres-
sure (1500 psi) at 100 ◦ C with a flush volume of 60% and 4
static cycles (static time of 8 min/cycle), sequentially using HPLC-
grade n-hexane, ethyl acetate and methanol. The n-hexane extract
(15.2 g, 7% yield) (92.9% inhibition of Mycobacterium tuberculosis at
100 ␮g/mL) was fractionated using VLC, to yield sixteen fractions of
increasing polarity (F1–F16). Each fraction was tested for activity
against Mycobacterium tuberculosis H37 Rv. Active fraction F1 was
identified as the pure compound 1 (600 mg). Two other active frac-
tions, F5 and F6, were combined and a portion (1.5 g) was further
subjected to gel filtration to afford ten sub-fractions (F5.1–F5.10).
Sub-fraction F5.9 was identified as compound 2 (80.3 mg).
The dried powdered aerial parts of Juniperus communis (740 g)
were Soxhlet extracted successively with n-hexane, ethyl acetate
and methanol. A portion of the n-hexane extract (16.4 g) (MIC of
64 ␮g/mL against Mycobacterium aurum) was subjected to VLC to
yield fourteen fractions of increasing polarity (F1–F14). Each frac-
tion was tested for activity against Mycobacterium aurum. Active
fraction F5 (3.5 g) was further fractionated by gel filtration to yield
seven sub-fractions (F5.1–F5.7). Active sub-fraction F5.4 was iden-
tified as compound 3 (1.1 g).
2.3.1. Longifolene (1)
Obtained as a pale yellow oil; [␣]D , IR, MS, 1 H and 13 C NMR iden-
tical with literature data (Lei and Fallis, 1993) and with an authentic
standard.
2.3.2. Totarol (2)
Obtained as a white amorphous solid; [␣]D , IR, MS, 1 H and 13 C
NMR identical with literature data (Ying and Kubo, 1991) and with
an authentic standard.
2.3.3. Trans-communic acid (3)
Obtained as a white amorphous solid; [␣]D , IR and MS identi-
cal with literature data (Muhammad et al., 1995). 1 H NMR (CDCl3 ,
400 MHz) (ı): 1.13 m and 1.87 m (H-1a/b), 1.53 m (H-2a/b), 1.05
m and 2.15 m (H-3a/b), 1.34 dd (J = 3.2, 11.7 Hz) (H-5), 1.96 m (H-
6a/b), 1.91 m and 2.39 m (H-7a/b), 1.74 s (H-9), 2.13 m and 2.36 m
(H-11a/b), 5.40 t (J = 6.6 Hz) (H-12), 6.32 dd (J = 17.4, 10.8 Hz) (H-14),
4.87 d (J = 10.6 Hz) and 5.03 d (J = 17.6 Hz) (H-15a/b), 1.74 s (H-16),
4.46 s and 4.83 s (H-17a/b), 1.24 s (H-18), 0.64 s (H-20); 13 C NMR
(CDCl3 , 100 MHz) (ı): 39.2 (C-1), 19.9 (C-2), 37.9 (C-3), 44.2 (C-4),
56.2 (C-5), 25.8 (C-6), 38.5 (C-7), 147.9 (C-8), 56.4 (C-9), 40.4 (C-10),
502 A.Y. Gordien et al. / Journal of Ethnopharmacology 126 (2009) 500–505
23.3 (C-11), 133.9 (C-12), 133.3 (C-13), 141.6 (C-14), 109.9 (C-15),
11.9 (C-16), 107.7 (C-17), 29.1 (C-18), 184.3 (C-19), 12.9 (C-20).
2.4. Determination of in vitro activity against non-tuberculous
mycobacteria
The activity against Mycobacterium aurum A+ (CIP 10482, Pas-
teur Institute, Paris, France), Mycobacterium phlei (NCTC 8151),
Mycobacterium fortuitum (NCTC 10394) and Mycobacterium smeg-
matis (NCTC 10265) was evaluated using a modification of a
previously reported procedure (Seidel and Taylor, 2004). Cultures
were incubated at 37 ◦ C on Columbia agar slants (supplemented
with 5% horse blood) and sub-cultured once at 37 ◦ C for 3 days
prior to an assay. Colonies were transferred to normal saline in
a vial containing some glass beads (425–600 ␮m) (Sigma–Aldrich,
UK). Bacterial suspensions were mixed vigorously, to disrupt vis-
ible clumps, and were left to settle for 5 min. Supernatants were
diluted in normal saline to match the turbidity of a McFar-
land 0.5 standard. Aliquots (50 ␮L) were then transferred to
cation-adjusted Mueller–Hinton broth (10 mL) (Sensititre® , Trek-
Diagnostics Systems, UK) to obtain the desired inoculum. Screening
was performed in microplates containing 100 ␮L of cation-adjusted
Mueller–Hinton broth in each well. Stock solutions of extracts were
prepared in a suitable solvent (DMSO, DMF, methanol or water) and
100 ␮L was serially twofold diluted across the plate in broth. The
bacterial inoculum (100 ␮L) was subsequently added to give about
2.5 × 105 CFU/mL in all wells. No deleterious effects were observed
at the highest concentration of solvents used (1.5%, v/v). Isoni-
azid, ethambutol and rifampicin (Sigma–Aldrich, UK) were used as
positive controls. The plates were incubated at 37 ◦ C for 5 days.
Inhibition of growth was detected following addition (20 ␮L) of
a methanolic solution (5 mg/mL) of MTT (Lancaster, UK) followed
by incubation at 37 ◦ C for 1 h. Viable bacteria reduced the yellow
dye to a purple colour. The MIC was defined as the lowest sample
concentration that prevented this change and exhibited complete
inhibition of macroscopic growth. Each sample was tested in dupli-
cate and on two separate days.
2.5. Determination of in vitro activity against Mycobacterium
tuberculosis H37 Rv and drug-resistant variants
The activity against Mycobacterium tuberculosis H37 Rv (ATCC
27294), H37 Rv-rifampicin-resistant (ATCC 35838), H37 Rv-
isoniazid-resistant (ATCC 35822), H37 Rv-streptomycin-resistant
(ATCC 35820) and a moxifloxacin-resistant isolate (generated
from H37 Rv at the University of Illinois at Chicago with an aspartic
acid to asparagine mutation in codon 94) was assessed according
to a method based on the microplate Alamar Blue assay (MABA)
(Collins and Franzblau, 1997). Briefly, stock solutions of sam-
ples were prepared in DMSO and added, at a concentration of
100 ␮g/mL, to two wells of a microplate containing Middlebrook
7H12 broth (Falzari et al., 2005). One well was inoculated with
broth containing 2 × 104 CFU Mycobacterium tuberculosis H37 Rv.
The second well received only media in order to assess background
fluorescence. Isoniazid, rifampicin, moxifloxacin and PA-824 (a
nitroimidazopyran-derived experimental antitubercular drug
candidate) were included as standard controls. Additional controls
consisted of bacteria + DMSO and media only. For MICs, twofold
serial dilutions were performed in 7H12 media. Each microplate
was incubated for 5 days at 37 ◦ C in a 5% CO2 atmosphere in a sealed
plastic bag. After incubation, one control growth was developed
with a mixture of Alamar blue solution (20 ␮L) (Trek Diagnostics,
Westlake OH, USA) and sterile 10% Tween 80 (12 ␮L). The plates
were re-incubated at 37 ◦ C for 24 h. After this, if the well turned
pink, the dye mixture was placed into all wells and the plates were
re-incubated for an additional 24 h. The mean fluorescence units
(FU) of media-only wells were subtracted from all other wells.
Results were expressed in terms of % inhibition defined as 1 − (test
well FU/mean FU of triplicate bacteria-only wells) × 100. The MIC
was defined as the lowest concentration effecting a reduction in
fluorescence of ≥90% relative to bacteria-only controls. Rifampicin,
isoniazid, moxifloxacin and PA-824 (Global Alliance for TB Drug
Development) were included as positive controls. Each sample
was assayed in duplicate.
2.6. Cytotoxicity studies
Pure compounds, isolated through the bioassay-guided frac-
tionation process, were assessed for cytotoxicity against mam-
malian Vero cells (ATCC CCL-81) using a cell proliferation assay
previously described (Falzari et al., 2005). Results were expressed
as the concentration effecting 50% inhibition of the growth of Vero
cells (IC50 ). Selectivity indices were calculated for each compound
by dividing the IC50 value with the MIC value.
2.7. Determination of in vitro activity against non-replicating
persistent Mycobacterium tuberculosis H37 Rv
The potential of samples to target the subpopulation of Mycobac-
terium tuberculosis in the non-replicating persistent (NRP) state
was assessed using a low oxygen recovery assay (LORA) (Cho
et al., 2007). This employed Mycobacterium tuberculosis H37 Rv
(pFCA-luxAB) which synthesises luciferase when actively grow-
ing (Changsen et al., 2003; Snewin et al., 1999). The strain was
cultured in 300 mL of Dubos Tween–albumin broth (supplier) in
a BioStatQ fermentor with a head space ratio of 0.5 and agitated at
120 rpm with no detectable perturbation of the medium surface.
The dissolved oxygen concentration (DOC) was monitored with an
Ingold oxygen sensor probe. Cells were harvested when the OD at
570 nm indicated achievement of the desired growth phase (i.e. late
non-replicating persistence). Aliquots of bacterial culture (50 mL)
were centrifuged at 2700 × g for 30 min, washed once with phos-
phate buffered saline (PBS), suspended in PBS (1 mL), and stored at
−80 ◦ C. Prior to use, cultures were thawed, diluted in Middlebrook
7H12 broth and sonicated for 15 s. Cultures were diluted to obtain
an OD at 570 nm of 0.03–0.05 and 2000–5000 relative light units
(RLU) per 100 ␮L (i.e. 5 × 105 to ∼2 × 106 CFU/mL). Twofold serial
dilutions of compounds were prepared in 100 ␮L in black 96-well
plates, and 100 ␮L of the cell suspension was subsequently added.
The microplate cultures were placed under anaerobic conditions
(oxygen < 0.16%) using an Anoxomat Model WS-8080, three cycles
of evacuation and filling with a mixture of 10% H2 , 5% CO2 , balance
N2 . Plates were incubated at 37 ◦ C for 7 days and then transferred
to an ambient gaseous condition (5% CO2 -enriched air) incubator
for a 24 h “recovery”. On day 7 and 8, 100 ␮L of culture were trans-
ferred to white opaque 96-well plates. A 10% solution of n-decanal
in ethanol was freshly diluted 10-fold in PBS and 100 ␮L added
to each well with an auto-injector. Luminescence was measured
in a Victor2 multi-label reader (1 s reading time). Samples reduc-
ing viability under these non-growth conditions led to a decreased
luciferase signal following aerobic recovery. Pure compounds were
tested at 50 and 10 ␮g/mL and MICs were determined.
3. Results and discussion
Upon fractionation of the n-hexane extract of Juniperus com-
munis roots, three active primary fractions were obtained, namely
F1, F5 and F6. Fraction F1 (97.7% inhibition of Mycobacterium
tuberculosis at 100 ␮g/mL) was identified as pure longifolene (1).
Further purification work on F5 and F6 (98.1 and 99.3% inhibition
of Mycobacterium tuberculosis at 100 ␮g/mL, respectively) afforded
one active secondary fraction, namely F5.9 (100% inhibition of
 A.Y. Gordien et al. / Journal of Ethnopharmacology 126 (2009) 500–505 503

Table 1
Activity of terpenoids isolated from Juniperus communis against Mycobacterium tuberculosis H37 Rv, non-replicating and drug-resistant variants.

Compound MIC values in ␮g/mL (␮M)

H37 Rv RIF-resistanta INH-resistanta SM-resistanta MOX-resistanta Non-replicating H37 Rv

(pFCA-luxAB)

Longifolene (1) 92.4 (452.2) 4.9 (24.0) 22.7 (111.1) 93.8 (459.0) 76.6 (374.8) 93.4 (457.1)
Totarol (2) 21.1 (73.7) 5.8 (20.2) 11.0 (38.4) 23.9 (83.4) 17.2 (60.0) 23.3 (81.3)
Trans-communic acid (3) >100 (>330.6) NDb NDb NDb NDb NDb
RIFa 0.05 (0.06) >3.29 (>4) 0.06 (0.07) 0.02 (0.03) 0.12 (0.15) 1.10 (1.34)
INHa 0.06 (0.41) 0.07 (0.48) >1.10 (>8) 0.07 (0.48) 0.11 (0.83) >17.5 (>128)
MOXa 0.28 (0.63) 0.11 (0.24) 0.14 (0.31) 0.18 (0.42) >7.01 (>16) >56 (>128)
PA-824a 0.14 (0.40) 0.04 (0.10) 0.17 (0.48) 0.04 (0.12) 0.11 (0.30) 0.95 (2.64)
a
RIF: rifampicin, INH: isoniazid, SM: streptomycin, MOX: moxifloxacin, PA-824: nitroimidazopyran derivative.
b
ND: not determined.

Mycobacterium tuberculosis at 100 ␮g/mL) which was identified
as totarol (2) (80.3 mg). Bioassay-guided fractionation of the n-
hexane extract of Juniperus communis aerial parts afforded one
active primary fraction, namely F5 (MIC of 16 ␮g/mL). The latter
was further purified to yield the active secondary fraction F5.4 (MIC
of 4 ␮g/mL), identified as trans-communic acid (3). The structure
elucidation of all compounds was carried out following the analysis
of their spectroscopic data, including extensive 1D- and 2D-NMR,
and by comparison with literature reports and commercial authen-
tic standards when available. The 1 H and 13 C NMR assignments for
compound 3 were identical with literature data (Smith et al., 2007),
except for C-19 which differed, and for H1/C1 and H3/C3 resonances
which were reversed.
Results of the investigation of the activity of pure isolated
compounds against Mycobacterium tuberculosis H37 Rv, and four
drug-resistant variants, using the microplate Alamar blue assay
(MABA) are presented in Table 1. It was observed that compound 2
with an MIC of 73.7 ␮M, showed the best activity against Mycobac-
terium tuberculosis H37 Rv. Compound 3 was not considered active
against Mycobacterium tuberculosis H37 Rv (MIC > 100 ␮g/mL). This
compound could not be tested against the drug-resistant strains
since it was found to have degraded at the time of the screening.
Compounds 1 and 2 were most active against the rifampicin-
resistant strain (MICs of 24 and 20.2 ␮M, respectively). Compound
2 was also most active against the isoniazid-, streptomycin-,
and moxifloxacin-resistant strains (MIC of 38.4, 83.4 and 60 ␮M,
respectively). Results of the investigation of the activity of pure
isolated compounds against non-replicating persistent Mycobac-
terium tuberculosis H37 Rv using the low oxygen recovery assay
(LORA) are also presented in Table 1. Compound 2 displayed the
best activity (MIC of 81.3 ␮M) in this assay.
Results of the investigation of the activity of pure isolated
compounds against non-tuberculous mycobacteria are reported
in Table 2. Compound 2 showed the best activity against all
species with MIC values in the range of 7–14 ␮M. Compound
3 showed good activity against Mycobacterium aurum but could
not be screened against other mycobacterial species for reasons
detailed above. Results of the cytotoxicity screening and calculated
selectivity indices (IC50 /MIC) of tested compounds are reported
in Table 3. Longifolene (1), totarol (2) and communic acid (3)
(Fig. 1) had IC50 values of 53.2 ␮g/mL (260.3 ␮M), 7.5 ␮g/mL and
40 ␮g/mL (132.2 ␮M), respectively. The low selectivity indices
obtained (SI < 10) indicated that the isolated terpenoids were rela-
tively toxic towards mammalian cells, especially when compared
to the high selectivity of the antibiotic control.
Juniperus communis (roots and bark) is a plant reputed to
be effective in the treatment of tuberculosis (Atkinson, 2003;
McCutcheon et al., 1997; UK CropNet MPNADB, 2009) and several
studies have reported the activity of extracts, particularly against
Mycobacterium tuberculosis (including some drug-resistant strains),
Mycobacterium avium, Mycobacterium aurum and Mycobacterium
smegmatis (Fitzpatrick, 1954; Grange and Davey, 1990; Jimenez-
Arellanes et al., 2003; McCutcheon et al., 1997; Newton et al.,
2002). Whilst several terpenoids have been identified as responsi-
ble for the antimycobacterial activity of Juniperus procera (another
species traditionally used as an anti-TB remedy) (Mossa et al., 2004;
Muhammad et al., 1995), the antimycobacterial activity of common
juniper has never been attributed to any pure active substance(s).
In our in vitro screening assays, the antitubercular activity of
Juniperus communis roots was attributed to the sesquiterpene longi-
folene (1) and the diterpene totarol (2). Longifolene has previously
been detected in Juniperus communis subsp. alpina root essential oil
(Gonny et al., 2006) and has been reported from Juniperus commu-
nis stem bark (Arya, 1962). This is, however, the first report of its
isolation from the roots and the first report of its antimycobacterial
activity. Totarol has previously been isolated from Juniperus procera
and other plants within the Cuppressaceae family (Constantine et
al., 2001; Iwamoto et al., 2001; Muhammad et al., 1995; Sharp et
al., 2001). It has recently been detected in some commercial gin
samples aromatised with Juniperus communis berries (Vichi et al.,
2008). Its isolation from the roots has never been reported. Totarol
is a known antimycobacterial agent with activity against Mycobac-

Table 2
Activity of terpenoids isolated from Juniperus communis against non-tuberculous mycobacteria.

Compound MIC values in ␮g/mL (␮M)

Mycobacterium aurum Mycobacterium fortuitum Mycobacterium phlei Mycobacterium smegmatis

Longifolene (1) 128 (626.4) >128 (>628.4) >128 (>628.4) >128 (>628.4)
Totarol (2) 2 (7.0) 4 (14.0) 4 (14.0) 2 (7.0)
Trans-communic acid (3) 4 (13.2) NDa NDa NDa
EMBb 2 (9.79) NDa NDa NDa
RIFb 1 (1.22) NDa NDa NDa
INHb 0.125 (0.91) NDa NDa 2 (14.58)
CIPb NDa 1 (3.02) 1 (3.02) NDa
DOXb NDa . NDa 1 (2.25) NDa
a
ND: not determined.
b
EMB: ethambutol; RIF: rifampicin; INH: isoniazid; CIP: ciprofloxacin; DOX: doxycycline.
504 A.Y. Gordien et al. / Journal of Ethnopharmacology 126 (2009) 500–505

Table 3
Cytotoxicity (IC50 ) and selectivity indices (IC50 /MIC) of terpenoids isolated from Juniperus communis.

Compound Longifolene (1) Totarol (2) Trans-communic acid (3) RIFa

IC50 in ␮g/mL (␮M) 53.2 (260.3) 7.5 (26.2) 40 (132.2) 101.2 (123)

Selectivity indices for

H37 Rv (MABA) 0.58 0.36 <0.40 2049
H37 Rv (LORA) 0.57 0.32 NDb 91.7
Mycobacterium aurum 0.42 3.75 NDb 101
Mycobacterium fortuitum <0.42 1.88 NDb NDb
Mycobacterium phlei <0.42 1.88 NDb NDb
Mycobacterium smegmatis <0.42 3.75 NDb NDb
a
RIF: rifampicin.
b
ND: not determined.

terium tuberculosis H37 Rv and some NTM species (Constantine et
al., 2001; Mossa et al., 2004; Muhammad et al., 1995). Its activ-
ity against Mycobacterium aurum, Mycobacterium fortuitum and
Mycobacterium phlei is reported here for the first time.
In our in vitro screening assays, the activity of Juniperus com-
munis aerial parts against Mycobacterium aurum was attributed to
trans-communic acid (3). The latter has previously been isolated
from Juniperus communis berries (Arya et al., 1961) and is known
to possess antibacterial activity (Muhammad et al., 1995; Smith
et al., 2007). This is here the first report of its isolation from the
aerial parts and of its antimycobacterial activity. It is interesting to
note that, in our assay, trans-communic acid was inactive against
Mycobacterium tuberculosis whilst the literature indicates that a
structurally related labdane diterpene (juniperexelsic acid) from
Juniperus procera has good activity against Mycobacterium tubercu-
losis (Topçu et al., 1999). This suggests that a 13,16 double bond
and/or a 3-O-acetylated position on the labdane skeleton may be
required for activity.
When the isolated terpenoids were screened for activity against
a panel of drug-resistant strains and against non-replicating per-
sistent Mycobacterium tuberculosis H37 Rv, none showed activity to
the same level as the standard antibiotics. Nevertheless, the effect
of longifolene (1) and totarol (2) on these drug-resistant and non-
replicating variants is reported here for the first time.
In conclusion, our study provides, at least in part, some scientific
basis for the ethnomedicinal use of common juniper as a traditional
anti-TB remedy. It is most likely that other compounds contribute
to the overall antitubercular effect observed in indigenous popu-
lations (e.g. through immunostimulation). The isolation of some
antimycobacterial substances from this plant, which thrive best in
wet ericaceous soils, confirmed our initial assumption that active
metabolites could be produced as defensive agents in response to
the predominance of mycobacteria in the environment. This sug-
gests that ericaceous plants and conifers may represent a promising
source of antimycobacterial agents, which merits further investi-
gation. The moderate activity and low selectivity of the terpenoids
tested in this study preclude their use as anti-TB drugs. However,
it is possible that, as previous studies have indicated that was the
case for totarol in combination with isoniazid (Mossa et al., 2004),
these compounds may prove to be good potentiators of the activ-
ity of conventional antitubercular drugs. They may also, following
optimisation, serve as templates for the development of anti-TB
agents in the future.

Acknowledgements

This work was supported by grant EP/D504988/1 from the

British Engineering and Physical Sciences Research Council (EPSRC-
Life Sciences Interface programme). The authors are thankful to
Rodney Shearer (Alba Trees Plc nursery) for the provision of plant
material and to Baojie Wan and Yuehong Wang (Institute for
Tuberculosis Research) for performing the antitubercular and cyto-
toxicity assays.

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