Antimycobacterial activity of ferutinin alone and in combination with antitubercular drugs against a rapidly growing surrogate of Mycobacterium tuberculosis

Article abstract of DOI:10.1080/14786419.2010.481623

The aim of this study was to investigate the antimycobacterial activity of the major daucane constituent, ferutinin (jaeschkeandiol p-hydroxybenzoate, 1), four of its natural analogues, its hydrolysis products, as well as methyl p-hydroxybenzoate (methylparaben) against Mycobacterium smegmatis, a rapidly growing surrogate of Mycobacterium tuberculosis. The agar dilution assay was utilised for an antimycobacterial evaluation of single compounds. A modified agar dilution assay, the checkerboard method, was utilised for evaluating the potentiating effect of 1 on different antitubercular drugs, namely isoniazid, ethionamide, rifampin and streptomycin. In the agar dilution assay, 1 exhibited higher potency (minimum inhibitory concentration [MIC] 10μgmL^-1) than streptomycin and rifampin (MIC 20 μg mL^-1 for each). Of the natural analogues, 8,9-epoxyjaeschkeandiol phydroxybenzoate and 8,9-epoxyjaeschkeandiol benzoate exhibited marginal activity (MIC ≥ 40 and 80μgmL^-1, respectively). The checkerboard method showed that the combination of 1 with each antitubercular drug led to mutual enhancement of the antimycobacterial activity with isoniazid and ethionamide, while no such effect was observed with rifampin or streptomycin. Based on this study and earlier studies with Staphylococcus aureus, the major constituent 1 may be responsible for the major part of the antimicrobial activity of the root of Ferula hermonis.

Full text of DOI:10.1080/14786419.2010.481623

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Antimycobacterial activity of ferutinin
alone and in combination with
antitubercular drugs against a rapidly
growing surrogate of Mycobacterium
tuberculosis
Ehab A. Abourashed a , Ahmed M. Galal b & Atef M. Shibl c
a College of Pharmacy, Chicago State University, 9501 South King
Drive, Chicago, IL 60628, USA
b National Center for Natural Products Research, School of
Pharmacy, University of Mississippi, Oxford, MS 38677, USA
c College of Pharmacy, King Saud University, Riyadh 11451, Saudi
Arabia
Version of record first published: 25 Mar 2011.
To cite this article: Ehab A. Abourashed , Ahmed M. Galal & Atef M. Shibl (2011):
Antimycobacterial activity of ferutinin alone and in combination with antitubercular drugs against
a rapidly growing surrogate of Mycobacterium tuberculosis , Natural Product Research: Formerly
Natural Product Letters, 25:12, 1142-1149
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Natural Product Research
Vol. 25, No. 12, July 2011, 1142–1149
Antimycobacterial activity of ferutinin alone and in combination with
antitubercular drugs against a rapidly growing surrogate of
Mycobacterium tuberculosis
a
Ehab A. Abourashed , Ahmed M. Galal and Atef M. Shibl
b
c
*
aCollege of Pharmacy, Chicago State University, 9501 South King Drive, Chicago,
IL 60628, USA; National Center for Natural Products Research, School of Pharmacy,
b
c
University of Mississippi, Oxford, MS 38677, USA; College of Pharmacy, King Saud
University, Riyadh 11451, Saudi Arabia
(Received 19 November 2009; final version received 21 March 2010)
The aim of this study was to investigate the antimycobacterial activity of
the major daucane constituent, ferutinin (jaeschkeandiol p-hydroxybenzo-
ate, 1), four of its natural analogues, its hydrolysis products, as well as
methyl p-hydroxybenzoate (methylparaben) against Mycobacterium smeg-
matis, a rapidly growing surrogate of Mycobacterium tuberculosis. The agar
dilution assay was utilised for an antimycobacterial evaluation of single
compounds. A modified agar dilution assay, the checkerboard method, was
utilised for evaluating the potentiating effect of 1 on different antituber-
cular drugs, namely isoniazid, ethionamide, rifampin and streptomycin. In
the agar dilution assay, 1 exhibited higher potency (minimum inhibitory
concentration [MIC] 10 mg mLꢀ1) than streptomycin and rifampin (MIC
20 mg mLꢀ1 for each). Of the natural analogues, 8,9-epoxyjaeschkeandiol p-
hydroxybenzoate and 8,9-epoxyjaeschkeandiol benzoate exhibited mar-
ginal activity (MIC ꢁ 40 and 80 mg mLꢀ1, respectively). The checkerboard
method showed that the combination of 1 with each antitubercular drug led
to mutual enhancement of the antimycobacterial activity with isoniazid and
ethionamide, while no such effect was observed with rifampin or strepto-
mycin. Based on this study and earlier studies with Staphylococcus aureus,
the major constituent 1 may be responsible for the major part of the
antimicrobial activity of the root of Ferula hermonis.
Keywords: Shirsh El Zallouh; Ferula hermonis; ferutinin; antimycobacter-
ial; rapidly growing mycobacteria; potentiation; antitubercular drugs
1. Introduction
The resistance of pathogenic microorganisms to antimicrobial drugs has increased
worldwide in the past two decades. This problem is specifically associated with the
antibiotics used for the treatment of infections caused by methicillin-resistant
Staphylococcus aureus (MRSA), Mycobacterium tuberculosis and fungal infections.
The resistance to antimicrobial agents necessitates increasing the dose of antibiotics
to high levels to attain therapeutic effect. The latter practice may result in the
*Corresponding author. Email: eabouras@csu.edu
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2011 Taylor & Francis
DOI: 10.1080/14786419.2010.481623
http://www.informaworld.com

Natural Product Research
1143
increased incidence of adverse effects and/or toxicity (Plorde, 1994). Excessive use of
antibiotics is also becoming a major public health concern due to the additional costs
of prescriptions, hospitalisation and increased rates of morbidity and mortality from
infections. To illustrate, community-acquired MRSA infections and the numbers of
MRSA isolated from blood and cerebrospinal fluid (CSF) samples have significantly
risen over the last decade (Dirk, 2007; Nakamura et al., 2008). Similarly, the number
of infections with vancomycin-resistant Enterococci, which were rare up until 1992,
has also increased. Most of the reasons for this increase are accounted for by
Enterococcus faecium, which had a 30% prevalence of resistance in 2000 (Mascini &
Bonten, 2005).
A fairly large number of plant natural products have been reported to exhibit
antimicrobial activity of experimental promise rather than clinical potential.
Antibiotic potentiation through synergistic interaction with natural products has
been investigated as an approach towards the minimisation of side effects of, and
resistance to, antibiotics (Didry, Dubreuil, & Pinkas, 1993; Gibbons, Oluwatuyi,
Veitch, & Gray, 2003; Kubo, Muroi, & Himejima, 1993; Park, Kim, & Hahm, 2004;
Periago, Palop, & Fernandez, 2001; Shin & Kang, 2003), especially when the natural
products are not sufficiently potent as single entities. Recently, peptides derived from
a Helicobacter pylori protein resulted in a four-fold reduction in the minimum
bactericidal concentrations (MBCs) of chloramphenicol against selected Gram-
positive and Gram-negative bacteria (Park et al., 2004). Diterpenes from Lycopus
europaeus had no antibacterial activity against the resistant strains of S. aureus, but
resulted in a two-fold potentiation of the activities of tetracycline and erythromycin
against the same staphylococcal strains (Gibbons et al., 2003).
Systemic infections caused by rapidly growing mycobacteria (RGMs),
Mycobacterium avium complex (MAC) and fungi, such as Cryptococcus neoformans
and Candida albicans, are serious conditions occurring primarily in immunocom-
promised neutropenic patients, especially those with AIDS (Iseman, 1999). For such
conditions, drug combinations that are periodically altered are routinely used in an
attempt to achieve maximum efficacy while reducing the side effects and slowing the
rate of the development of resistance (Rex, Sobel, & Powderly, 1999). Isonicotinic
acid hydrazide (isoniazid, INH), streptomycin and isoniazrifampinid arefrontline
drugs used in the combined treatment of M. tuberculosis (with MICs 0.01–
0.25 mg mLꢀ1). However, the first two are not very effective against other
mycobacteria (MICs41 mg mLꢀ1
; Lemke, 2002). Two more recent reports
(Abourashed, Galal, Shibl, & Mossa, 2007; Mossa, El-Feraly, & Muhammad,
2004) showed that the constituents from Juniperus procera, Ferula communis,
Plumbago zeylanica, Syzygium aromaticum and Aframomum melegueta exhibited an
in vitro synergistic activity with INH against four atypical mycobacteria from the
RGM group and against M. tuberculosis (TB).
The root of Ferula hermonis Boiss (family Apiaceae), known in Syria, Lebanon,
and in the Middle East as ‘Shirsh El Zallouh’, has been used in local traditional
medicine as an aphrodisiac for male erectile dysfunction and as a general tonic.
Zallouh is also believed to possess antimicrobial and antioxidant activities
(Abourashed, Galal, El-Feraly, & Khan, 2001; Fitzsimmons, 2009; Hilan et al.,
2007). Ferutinin (1) is a major daucane sesquiterpene ester, isolated from F. hermonis
and other species of Ferula (Abourashed et al., 2001; Galal, 2000). Ferutinin
exhibited antimicrobial activity against S. aureus (MIC 3.5 mg mLꢀ1) and a weak

1144
E.A. Abourashed et al.
antifungal effect against Aspergillus niger (Galal et al., 2001). Other natural
analogues of 1 include jaeschkeandiol benzoate (2), jaeschkeandiol vanillate (3),
epoxyferutinin (4), epoxyjaeschkeandiol benzoate (5), and others (Diab, Dolmazon,
& Bessiere, 2001; Diab, Dolmazon, & Fenet, 2001; Lhuillier et al., 2005). Since none
of 1 or its natural analogues (2–5) have been tested against Mycobacteria, the
purpose of this work was to investigate the antimycobacterial effect of compounds
1–5, the hydrolysis products of 1 (6, jaeschkeandiol; 7, p-hydroxybenzoic acid), as
well as methyl p-hydroxybenzoate (8, methylparaben) in an in vitro assay utilising
Mycobacterium smegmatis as a representative of RGMs and as a surrogate
microorganism to TB. The potentiating and synergistic effects of 1 on a number
of antitubercular drugs were also investigated in the same microorganism, and the
results are presented in this article.
O
1
8
4
6
HO
HO
R1
H
H
O
O
R
R
O
O
Epoxyferutinin, 4
R=OH
Ferutinin, 1
Jaeschkeanadiol benzoate, 2 R=H, R1=H
R=OH, R1=H
Epoxyjaeschkeanadiol benzoate, 5 R=H
Jaeschkeanadiol vanillate, 3
R=OH, R1=OMe
COOR
S
NHNH2
NH2
O
HO
H
Me
OH
N
N
OH
Ethionamide
Isoniazid (INH)
7
Jaeschkeanadiol, 6 R = H, p-Hydroxybenzoic acid,
R = CH3, Methylparaben, 8
OH
NH
NH
HO
H2N
NH2
H3COCO
HN
HO
HN
OH
OH
O
O
OH
OH HN
O
N
OCH3
CHO
N
O
OH
NHCH3
O
O
OH
OH
OH
N
O
O
OH
Streptomycin
Rifampin
2. Results
2.1. Antimycobacterial activity of individual compounds
In the agar dilution technique, 1 was the most active antimycobacterial (against
M. smegmatis) of all the tested compounds, with an MIC of 10 mg mLꢀ1. The MIC of
1 was even lower than those of streptomycin and rifampin (MICs 20 mg mLꢀ1 for
each). All other compounds were either inactive or marginally active, such as 4 and 5

Natural Product Research
1145
Table 1. Checkerboard chartsa and FIC indices for ferutinin in combination with
(A) isoniazid and (B) ethionamide.
Ferutinin (mg mLꢀ1
)
5
2.5
1.25
0.625
Panel A: isoniazid (FIC index ¼ 0.250)
Isoniazid (mg mLꢀ1
)
10
5
þ
þ
þ
ꢀ
þ
þ
þ
ꢀ
þ
þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2.5
1.25
Panel B: ethionamide (FIC index ¼ 0.125)
Ethionamide (mg mLꢀ1
)
10
5
þ
þ
þ
þ
þ
þ
þ
ꢀ
þ
þ
ꢀ
ꢀ
þ
ꢀ
ꢀ
ꢀ
2.5
1.25
a
Note: Highest concentration of each tested compound ¼ ½MIC.
(MICs ꢁ 40 and 80 mg mLꢀ1, respectively). The hydrolysis products of 1 were also
inactive.
2.2. Potentiation of antimycobacterial activity
Based on its significant antimycobacterial activity, 1 was selected for the potentiation
assays in combination with each of the known antitubercular agents: streptomycin,
rifampin, ethionamide and isoniazid. Strong antimycobacterial synergism
(FIC index50.50) was exhibited between 1 and both ethionamide and isoniazid
(Table 1). At 1.25 mg mLꢀ1 (1/8 MIC), 1 increased the antimycobacterial activity of
isoniazid eight-fold, with an FIC index of 0.25 (Panel A). Similarly, at 5 mg mLꢀ1 (1/2
MIC), 1 increased the antimycobacterial activity of ethionamide 16-fold, with an
FIC index of 0.125 (Panel B). No potentiation was observed between 1 and either
streptomycin or rifampin.
3. Discussion
Comparison of the antimycobacterial effect of the natural esters and the hydrolysis
products of 1 showed that the p-hydroxybenzoyl moiety was a prerequisite for
activity and that 1 was the most active of the natural esters. Thus, 1 was chosen for
further evaluation as a potentiating agent. It is interesting to note that
p-hydroxybenzoic acid, which is commonly used as a starting material for
preparation of cosmetic preservatives (parabens), in addition to its methyl ester
(methylparaben), did not display any antimycobacterial activity against
M. smegmatis. Also, the high MIC values observed for rifampin and streptomycin
may be attributed to the reported natural resistance of M. smegmatis towards
rifampin, which may also be applicable to streptomycin (Hetherington, Watson, &
Patrick, 1995; Quan, Venter, & Dabbs, 1997).

1146
E.A. Abourashed et al.
The synergistic effect between 1 and both isoniazid and ethionamide, with FIC
indices of 0.25 and 0.125, respectively, correlates with our previously reported
synergism between paradol, a natural phenolic ketone, and isoniazid (Abourashed
et al., 2007; Galal, 1996). The observed potentiation may be due to an enhancing
effect on the permeability of the mycobacterial cell membrane to antimycobacterial
drugs (Fitzgerlad et al., 2004). Since isoniazid and ethionamide are chemically
analogous, having a pyridine ring and an amide (acetamide and thionamide in
isoniazid and ethinoamide, respectively), it is rational to expect a similar trend in
their interaction with 1. The lack of synergism exhibited by ferutinin with
streptomycin, an aminocyclitol glycoside, and isoniazrifampinid, an ansamacrolide,
may be due to their relative bulkiness and structural dissimilarity as compared to
isoniazid and ethionamide. Thus, synergism based on enhanced permeability may
not be operational in this case.
The sporadic reports on the use of zallouh root as a traditional remedy for certain
infections in humans and animals may have some validity in view of our current
results with ferutinin, which is one of the major constituents of the root. The
antimicrobial indication of zallouh is also supported by earlier reports regarding the
activity of ferutinin against Gram-positive bacteria (Galal, 2000). The potentiating
and synergistic effects exhibited by ferutinin are a possible approach with which to
improve the safety and/or antimycobacterial efficacy of isoniazid and ethionamide,
as well as for reducing the development of microbial resistance to these drugs.
Further evaluation of ferutinin, and other small molecules, against an extended
spectrum of microorganisms and in appropriate animal models should be the subject
of further research.
4. Experimental
4.1. Plant material and chemicals
Roots of F. hermonis were purchased from the local herbal market in Syria in 2000
and were identified by Dr Sultan-ul-Abdeen, College of Pharmacy, King Saud
University, Saudi Arabia. A voucher specimen was deposited at the herbarium of the
college. Authenticity of the sample was later verified by TLC and HPLC
fingerprinting of the total methanolic extract of the powder (Abourashed et al.,
2001). The antitubercular drugs and m-chloroperbenzoic acid (MCPBA) were
purchased from Fluka AG (Buchs SG, Switzerland) and Sigma–Aldrich (St. Louis,
MO), respectively. Methylparaben was purchased from Sigma–Aldrich. Ferutinin
and the natural derivatives were isolated from the n-hexane extract of F. hermonis
roots.
4.2. Extraction and isolation
Isolation of the tested compounds followed a published procedure (Galal, 2000).
In brief, powdered root of F. hermonis (50 g) was exhaustively extracted with hot
n-hexanes, using a Soxhlet apparatus, and the extract was concentrated at 40ꢂC
under vacuum to an oily residue (4 g). This residue was partitioned between MeCN
and n-hexanes. The MeCN fraction was subjected to silica gel column chromatog-
raphy, eluted with mixtures of n-hexanes/EtOAc with increasing polarity, to isolate

Natural Product Research
1147
the target constituents. The structures of the isolated compounds were confirmed by
1H- and 13C-NMR spectroscopy methods. The purity of the compound was found to
be more than 98% by HPLC.
4.3. Hydrolysis of 1
Compound 1 was hydrolysed by refluxing with 10% KOH in MeOH for 2 h. The
liberated alcohol was back-extracted with ether followed by the removal of the
solvent to obtain jaeschkeanadiol (6, 74% yield). The aqueous phase was acidified
with 1N HCl and extracted with diethylether to afford p-hydroxybenzoic acid (7)
after evaporation of the solvent. The identities of 6 and 7 were confirmed by
comparing their NMR data with that in the literature.
4.4. Preparation of 4 and 5
The epoxy derivatives were prepared from the corresponding parent compounds by
epoxidation at D8,9 using MCPBA, following a procedure given in the literature
(Galal, 2000).
4.5. Evaluation of the antimycobacterial activity
The atypical mycobacteria, M. smegmatis (NCTC RR10), was obtained from the
National Collection of Type Culture, Central Public Health Laboratory, London,
UK. The agar serial dilution technique was utilised for determining the MIC values
of the tested compounds against the selected microorganisms (Mitscher, Leu,
Bathala, & Beal, 1972). Each compound was evaluated at different concentration
levels (200–10 mg mLꢀ1) by mixing decreasing volumes of a DMSO stock solution of
each compound with 10 mL of molten Mueller–Hinton agar (45–50ꢂC) to obtain the
final concentration. The inocula were prepared by growing the culture overnight in
Mueller–Hinton broth to yield a viable count of 109 cfu mLꢀ1. The culture was
diluted 1 : 1000 in a pre-warmed broth to give a final density of 104 cfu per spot. Test
organisms were streaked in a radial pattern and then the plates were incubated at
35ꢂC for 16–20 h. Complete suppression of growth was observed prior to declaring
the compound to be active at the respective concentration.
The checkerboard method for evaluating synergistic antimycobacterial effect was
employed to determine the MIC values of differently paired combinations of the
tested compounds (Beale & Sutherland, 1983; Mackay, Milne, & Gould, 2000). A
protocol similar to the one described for MIC determination was adopted with the
difference that serial two-fold dilutions of each compound were tested in combina-
tion with two-fold dilutions of another compound from the selected group. Each
evaluation was run in triplicate. The fractional inhibitory concentration (FIC)
indices were calculated from the following formula (Beale & Sutherland, 1983):
FIC index ¼ ðA=MICAÞ þ ðB=MICBÞ ¼ FICA þ FICB,
where A is the lowest inhibitory concentration of the first component of a binary
mixture in its row, MICA is the MIC of the component alone, B is the lowest
inhibitory concentration of the second component of the mixture in its row and

1148
E.A. Abourashed et al.
MICB is the MIC of the second component alone. Total synergism (FIC ꢃ 0.5),
partial synergism (0.55FIC ꢃ 0.75) and additive effect (0.755FIC ꢃ 2) or antago-
nism (FIC42) are the possible interpretations based on FIC index values (Didry
et al., 1993).
Acknowledgements
The authors are grateful to King Saud University College of Pharmacy Research Center for
supporting this project (grant no. CPRC 198). Appreciation is also due to Mr Mostafa Omar
Khaled for conducting the antimicrobial assays.
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