Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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4 Journal of Ethnopharmacology
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journal homepage: www.elsevier.com/locate/jep
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Research Paper
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Antitubercular activity of Arctium lappa and Tussilago farfara extracts
13 and constituents
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15 Jinlian Zhao a, Dimitrios Evangelopoulos b,1, Sanjib Bhakta b, Alexander I. Gray a,
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Q1 Véronique Seidel a,n
18 a
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK
b
19 Mycobacteria Research Laboratory, Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck, University of London,
London, UK
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23 art ic l e i nf o a b s t r a c t
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25 Article history: Q2 Ethnopharmacological relevance: Arctium lappa and Tussilago farfara (Asteraceae) are two plant species
Received 13 February 2014 used traditionally as antitubercular remedies. The aim of this study was (i) to screen Arctium lappa and
26 Received in revised form Tussilago farfara extracts for activity against Mycobacterium tuberculosis and (ii) to isolate and identify the
27 28 May 2014
compound(s) responsible for this reputed anti-TB effect.
28 Accepted 14 June 2014
Materials and methods: The activity of extracts and isolated compounds was determined against
29 Mycobacterium tuberculosis H37Rv using a high throughput spot culture growth inhibition (HT-
30 Keywords: SPOTi) assay.
31 Antitubercular activity Results: The n-hexane extracts of both plants, the ethyl acetate extract of Tussilago farfara and the
Arctium lappa
32 dichloromethane phase derived from the methanol extract of Arctium lappa displayed antitubercular
Tussilago farfara
33 activity (MIC 62.5 μg/mL). Further chemical investigation of Arctium lappa led to the isolation of
Mycobacterium tuberculosis
34 n-nonacosane (1), taraxasterol acetate (2), taraxasterol (3), a (1:1) mixture of β sitosterol/stigmasterol
35 (4), isololiolide (5), melitensin (6), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-
36 glucoside (10). Compounds isolated from Tussilago farfara were identified as a (1:1) mixture of
β sitosterol/stigmasterol (4), trans-caffeic acid (7), kaempferol (8), quercetin (9), kaempferol-3-O-gluco-
37
side (10), loliolide (11), a (4:1) mixture of p-coumaric acid/4-hydroxybenzoic acid (12), p-coumaric acid
38
(13). All compounds were identified following analyses of their physicochemical and spectroscopic data
39 (MS, 1H and 13C-NMR) and by comparison with published data. This is the first report of the isolation of
40 n-nonacosane (1), isololiolide (5), melitensin (6) and kaempferol-3-O-glucoside (10) from Arctium lappa,
41 and of loliolide (11) from Tussilago farfara. Amongst the isolated compounds, the best activity
42 was observed for p-coumaric acid (13) (MIC 31.3 μg/mL or 190.9 μM) alone and in mixture with
43 4-hydroxybenzoic acid (12) (MIC 62.5 μg/mL).
44 Conclusions: The above results provide for the first time some scientific evidence to support, to some
45 extent, the ethno-medicinal use of Arctium lappa and Tussilago farfara as traditional antitubercular
46 remedies.
& 2014 Published by Elsevier Ireland Ltd.
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52 1. Introduction treatment involves a long course of a combination of antibiotics
53 and is associated with poor patient compliance, which has led to
54 Tuberculosis (TB) is a major global health problem and cur- the emergence of multi-drug resistant (MDR) and extensively-
55 rently the leading bacterial killer worldwide. The World Health drug resistant (XDR) forms of TB (WHO, 2013). The treatment of
56 Organisation has estimated that Mycobacterium tuberculosis, the MDR-TB requires expensive second-line drugs and XDR-TB is often
57 causative agent of TB, was responsible for 8.6 million new cases incurable. There is now an urgent need to discover and develop
58 and 1.3 million deaths in 2012. The current recommended new anti-TB agents (Zumla et al., 2013). This has led to a renewed
59 interest in the screening and purification of novel anti-TB agents
60 n
from natural sources (Guzman et al., 2012; Santhosh and
Corresponding author. Tel.: þ 44 141 548 2751; fax: þ 44 141 552 2562.
61 Suriyanarayanan, 2014).
E-mail address: veronique.seidel@strath.ac.uk (V. Seidel).
62 1
Current address: Centre for Clinical Microbiology, Department of Infection, Arctium lappa (burdock) is a biennial herbaceous plant tradi-
63 Royal Free Campus, University College London, London, UK. tionally used for its diuretic, carminative, anti-inflammatory,
64 http://dx.doi.org/10.1016/j.jep.2014.06.034
65 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
66

Please cite this article as: Zhao, J., et al., Antitubercular activity of Arctium lappa and Tussilago farfara extracts and constituents. Journal
of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.034i
 2 J. Zhao et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 antiseptic and detoxifying properties (Kamkaen et al., 2006; Park (London, UK) in 2011. The plants were ground to a fine powder
2 et al., 2007). Burdock roots, seeds and leaves are also used as prior to extraction.
3 a traditional anti-TB medicine (Ritchason, 1995). Burdock is known
4 to contain some lignans (Ming et al., 2004; Kamkaen et al., 2006; 2.3. Extraction and isolation
5 Park et al., 2007), terpenoids (Costa et al., 1993; Tsuneki et al.,
6 2005), sterols (Ming et al., 2004; Mizushina et al., 2006), flavo- Arctium lappa (988 g) was Soxhlet extracted successively with
7 noids (Ferracane et al., 2010; Chen et al., 2011), hydroxycinnamic n-hexane (yield: 3% w/w), EtOAc (yield: 3% w/w) and MeOH (yield:
8 acid derivatives (Maruta et al., 1995; Ferracane et al., 2010) and 4% w/w). The n-hexane extract (30 g) was subjected to VLC eluting
9 polyacetylenes (Takasugi et al., 1987). Burdock extracts have anti- with hexane:EtOAc mixtures and then EtOAc:MeOH mixtures of
10 inflammatory (Lin et al., 1996) and anti-diabetic properties (Cao et increasing polarity. Fractions eluted with 2–10% EtOAc in n-hexane
11 al., 2012). Burdock leaf, fruit and root extracts display antimicro- were pooled (2.8 g) based on a similar TLC profile and subjected to
12 bial activity ( Pereira et al., 2005; Keyhanfar et al., 2011; He et al., gel filtration eluting with 5% n-hexane in dichloromethane fol-
13 2012 ). lowed by CC. Elution on CC with 0–5% EtOAc in hexane afforded 1
14 Tussilago farfara (coughwort) is a perennial herbaceous plant (156.9 mg). Elution with 10% EtOAc in hexane yielded 91 sub-
15 used to treat sore throats and lung ailments such as bronchitis, fractions (F1–F91). F1–F7 were combined and subjected to further
16 asthma and chronic cough including TB (Allen and Hatfield, 2004; CC eluting with 5% EtOAc in hexane to afford 2 (97.4 mg). F31–F37
17 Liu et al., 2006). It is also used for its detoxifying properties and for were combined and further subjected to gel filtration (5%
18 gout, skin, liver and kidney diseases (Darwin, 1996). Coughwort n-hexane in dichloromethane) to afford 3 (41.8 mg). F56–F91 were
19 contains sesquiterpene lactones (Liu et al., 2006; Park et al., 2008), pooled to afford 4 (43.3 mg). The EtOAc extract (30 g) was
20 triterpenes (Yaoita and Masao, 1998; Liu et al., 2006), flavonoids subjected to VLC eluting with hexane:EtOAc mixtures and then
21 (Liu et al., 2006), alkaloids (Luethy et al., 1980; Liu et al., 2006) and EtOAc:MeOH mixtures of increasing polarity. The MeOH extract
22 hydroxycinnamic acid derivatives (Liu et al., 2007). Coughwort (40 g) was partitioned between dichloromethane, n-butanol and
23 extracts have antimicrobial (Dulger and Gonuz, 2004; Turker and water. The dichloromethane phase (5.1 g) was subjected to VLC
24 Usta, 2008; Kačániová et al., 2013), anti-inflammatory (Benoit et eluting with hexane:EtOAc mixtures of increasing polarity. The
25 al., 1976; Jeong et al., 2013), cardiostimulatory (Li and Wang, 1988), fraction eluted with 50% EtOAc in hexane was subjected to gel
26 neuroprotective and anti-oxidative activity (Cho et al., 2005). To filtration in MeOH followed by CC eluting with 25% EtOAc in
27 the best of our knowledge, there has been no published report on hexane to yield 5 (8.9 mg). Further CC elution with 30% EtOAc in
28 the antitubercular screening of neither burdock nor coughwort hexane and subsequent gel filtration in EtOAc afforded 6
29 extracts. We report, herein, a chemical and biological investigation (25.5 mg). The butanol phase (13.5 g) was subjected to VLC eluting
30 on the aerial parts of burdock and coughwort for the presence of with hexane:EtOAc mixtures of increasing polarity. The fractions
31 compounds with whole-cell antitubercular activity. eluted with up to 10% MeOH in EtOAc were pooled and subjected
32 to gel filtration in MeOH and gave 7 (18 mg), 8 (1 mg) and 9
33 (3 mg). The fractions eluted with 15–25% MeOH in EtOAc were
34 pooled and subjected to CC. Elution with 10% MeOH in EtOAc
35 afforded 10 (18 mg).
2. Materials and methods
36 Tussilago farfara (995 g) was Soxhlet extracted successively
37 with n-hexane (yield: 2.3% w/w), EtOAc (yield: 1.4% w/w) and
2.1. General
38 MeOH (yield: 9.1% w/w). The n-hexane extract (23 g) was sub-
39 jected to VLC eluting with hexane:EtOAc mixtures of increasing
Solvents were from Fisher Scientific (UK) except for NMR
40 polarity. Gel filtration (5% n-hexane in dichloromethane) of the
deuterated (99.9%) solvents (Sigma-Aldrich, UK). TLC analysis
41 fractions eluted with 30% EtOAc in n-hexane and 80% EtOAc in
was carried out on silica gel 60 PF254 pre-coated plates (VWR
42 n-hexane yielded 4 (99.7 mg) and 11 (10.8 mg), respectively. The
International, UK). Compounds on TLC plates were detected under
43 EtOAc extract (14 g) was subjected to VLC eluting with hexane:
short (λ¼254 nm) UV light and visualised by spraying plates with
44 EtOAc mixtures and then EtOAc:MeOH mixtures of increasing
either p-anisaldehyde or vanillin sulphuric acid reagent, followed
45 polarity. Gel filtration (in MeOH) of the fraction eluted with 60%
by heating until coloured spots appeared. Silica gel 60H (VWR
46 EtOAc in n-hexane afforded 8 (42.2 mg), 12 (27.6 mg) and 13
International, UK) was used for vacuum liquid chromatography
47 (1 mg). Gel filtration (in MeOH) of the fraction eluted with 80%
(VLC). Open column chromatography (CC) was carried out using
48 EtOAc in n-hexane yielded 7 (1 mg) and 9 (4 mg). Gel filtration (in
silica gel 60 (mesh size, 0.063–0.200 mm) (VWR International, UK)
49 MeOH) of the fraction eluted with 20% MeOH in EtOAc gave 10
and gel filtration was performed using Sephadexs (LH-20-100)
50 (12.1 mg). The MeOH extract (91 g) was partitioned between
(Sigma-Aldrich, UK). 1H and 13C NMR experiments were carried
51 dichloromethane, n-butanol and water. All extracts and isolated
out on JEOL (JNM LA400) 400 MHz, Bruker 500 or 400 MHz
52 compounds were stored at  20 1C prior to testing.
instruments. Unless otherwise stated, HMBC experiments used
53
a time delay of 0.07 s (e.g. JCH ¼7 Hz). All spectra were referenced
54 2.4. Screening against Mycobacterium tuberculosis
on the residual solvent peaks and processed using Mestre Novas
55
(MNova) software version 8.0.0 (Mestrelab Research SL, Spain).
56 Antitubercular activity was determined in vitro against Myco-
High resolution electron impact (HREI) mass spectra were
57 bacterium tuberculosis H37Rv (ATCC27294) using a high-throughput
recorded on a JEOL 505HA spectrometer using direct probe at
58 version of the spot culture growth inhibition (HT-SPOTi) assay
elevated temperature (110–160 1C) at 70 eV. Positive ion and
59 (Evangelopoulos and Bhakta, 2010; Gupta and Bhakta, 2012;
negative ion mode ESI experiments were performed on a Thermo
60 Guzman et al., 2013). Briefly, Mycobacterium tuberculosis H37Rv
Finnigan LCQ-Deca Iontrap or Orbitrap HRESI mass spectrometer.
61 was initiated from a cryopreserved glycerol stock, passaged twice
62 for growth uniformity and grown at 37 1C in 10 mL Middlebrook
63 2.2. Plant material 7H9 liquid medium supplemented with 10% (v/v) oleic acid/albu-
64 min/dextrose/catalase (OADC) until the log phase (OD600E0.7).
65 Dried aerial parts of Arctium lappa L. (Asteraceae) and Tussilago Mycobacterial cultures were first checked for quality control using
66 farfara L. (Asteraceae) were purchased from G. Baldwin & Co Ltd. cold Ziehl–Neelsen staining. The mycobacterial suspension was

Please cite this article as: Zhao, J., et al., Antitubercular activity of Arctium lappa and Tussilago farfara extracts and constituents. Journal
of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.034i
 J. Zhao et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

1 then prepared by dilution to achieve 1  105 CFU/mL inoculum for Table 1

2 further use. Extracts/compounds were dissolved in DMSO. Serial Activity of Arctium lappa extracts and isolated compounds against Mycobacterium
tuberculosis H37Rv in the HT-SPOTi assay.
3 dilution was performed in a sterile, thin 96-well frosted subskirted
4 microtitre plate. A column containing only DMSO was also included Sample MICs μg/mL (μM)
5 as negative control. 2 μL of each of the diluted extracts/compounds
6 was then transferred into sterile 96-well plates. The plates were n-hexane extract 62.5
7 filled up to 200 μL with Middlebrook 7H10 agar medium enriched Ethyl acetate extract 125
Methanol extract/dichloromethane phase 62.5
8 with 10% (v/v) OADC using the MultidropCombi microplate dis- Methanol extract/butanol phase NAa
9 penser (Thermo-Fisher Scientific). Plates were dried and 2 μL Methanol extract/water phase NAa
10 prepared mycobacterial suspension was spotted (105 CFU/mL) at n-nonacosane (1) NAa
11 the centre of each well by either the use of a single pipettor or by Taraxasterol acetate (2) NAa
Taraxasterol (3) NAa
12 using the MultidropCombi. Then the plates were incubated in
β-sitosterol/stigmasterol (1:1) mix (4) 125
13 sealed bags at 37 1C for 2 weeks. MICs were determined as the Isololiolide (5) NAb
14 lowest concentration of extracts/compounds showing no visible Melitensin (6) NAb
15 mycobacterial growth. The first-line anti-TB drugs isoniazid and Isoniazid 0.01 (0.07)
16 rifampicin were included as antibiotic controls. Each sample was Rifampicin 0.01 (0.01)

17 assayed in two biological replicates. a

NA: no activity at 125 μg/mL.
18 b
NA: no activity at 500 μg/mL.
19
20 3. Results and discussion
21
22 Compounds isolated from Arctium lappa were identified as Table 2
23 n-nonacosane (1) (Chen et al., 2008 ), taraxasterol acetate (2) Activity of Tussilago farfara extracts and isolated compounds against Mycobacterium
24 (Khalilov et al., 2003), taraxasterol (3) (Mahato and Kundu, 1994), β tuberculosis H37Rv in the HT-SPOTi assay.
25 sitosterol/stigmasterol in a (1:1) mixture (4) (Wright et al., 1978),
Sample MICs μg/mL (μM)
26 isololiolide (5) (Kimura and Maki, 2002), melitensin (6)
27 (Medjroubi et al., 2003), trans-caffeic acid (7) (Durust et al., n-hexane extract 62.5
28 2001), kaempferol (8) (Xiao et al., 2006; Lee et al., 2009), quercetin Ethyl acetate extract 62.5
29 (9) (Xiao et al., 2006; Xiang et al., 2011), kaempferol-3-O-glucoside Methanol extract/dichloromethane phase 500
Methanol extract/butanol phase NAa
30 (10) (Xiao et al., 2006; Lee et al., 2009).
Methanol extract/water phase NAa
31 Compounds isolated from Tussilago farfara were identified as β-sitosterol/stigmasterol (1:1) mix (4) 125
32 β sitosterol/stigmasterol in a (1:1) mixture (4), trans-caffeic acid Caffeic acid (7) 250 (1387.7)
33 (7), kaempferol (8), quercetin (9), kaempferol-3-O-glucoside (10), Kaempferol (8) NAa
Quercetin (9) 500 (1654.3)
34 loliolide (11) (Kimura and Maki, 2002; Lee et al., 2009), p-coumaric
Kaempferol -3-O-glucoside (10) NAa
35 acid/4-hydroxybenzoic acid in a (4:1) mixture (12) (Ou et al., Loliolide (11) 250 (1273.9)
36 2011), p-coumaric acid (13) (Durust et al., 2001; Kuddus et al., p-coumaric acid/4-hydroxybenzoic acid (4:1) mix (12) 62.5
37 2010; Ou et al., 2011). All compounds were identified following p-coumaric acid (13) 31.3 (190.9)
38 analysis of their physicochemical and spectroscopic data (MS, 1H, Isoniazid 0.01 (0.07)
13 Rifampicin 0.01 (0.01)
39 C-NMR, COSY, NOESY, HMBC, HSQC) and by comparison with
40 published data. This is the first report of the isolation of n- a
NA: no activity at 500 μg/mL.
41 nonacosane (1), isololiolide (5), melitensin (6) and kaempferol-3-
42 O-glucoside (10) from Arctium lappa. Although kaempferol (8) has
43 previously been isolated from Arctium lappa roots (Chen et al.,
44 2011), this is the first report of its presence in the aerial parts. This wall and that their antimycobacterial activity depends on the type
45 is also the first report of the isolation of loliolide (11) from of substituents present on the phytyl moiety (Rugutt and Rugutt,
46 Tussilago farfara. 2002, 2012). To the best of our knowledge, this is the first time that
47 The results of the antitubercular screening of Arctium lappa n-nonacosane (1), taraxasterol acetate (2), isololiolide (5) and
48 extracts and selected compounds are reported in Table 1. Among melitensin (6) are screened for anti-TB activity.
49 the tested extracts, only the n-hexane extract and the dichlor- The results of the antitubercular screening of Tussilago farfara
50 omethane phase derived from the methanol extract displayed extracts and compounds are reported in Table 2. The n-hexane and
51 anti-TB activity (MIC 62.5 μg/mL). The ethyl acetate extract was ethyl acetate extracts showed the highest activity (MIC 62.5 μg/
52 moderately active at 125 μg/mL. Among the compounds isolated mL). The β-sitosterol/stigmasterol mixture (4) and loliolide (11)
53 from the n-hexane extract, only the β-sitosterol/stigmasterol isolated from the n-hexane extract were active at 125 and 250 μg/
54 mixture (4) revealed activity (MIC ¼125 μg/mL). Taraxasterol acet- mL, respectively, suggesting that the activity of the n-hexane
55 ate (2) and taraxasterol (3) were inactive at 125 μg/mL, whilst n- extract may be attributable in part to these compounds. This is
56 nonacosane (1), isololiolide (5) and melitensin (6) had no activity the first time that loliolide (11) is screened for anti-TB activity.
57 at 500 μg/mL. The activity observed for the n-hexane extract was Among the compounds isolated from Tussilago farfara ethyl acetate
58 stronger than that observed for isolated compounds, suggesting extract, quercetin (9) had an MIC of 500 μg/mL, whilst kaempferol
59 that the activity could either be attributable to other (non- (8) and kaempferol-3-O-glucoside (10) were both inactive at the
60 purified) phytochemical(s) or to compounds acting synergistically. highest concentration of 500 μg/mL. The best activity was
61 The activity of (4) in the HT-SPOTi assay correlated well with observed for p-coumaric acid (13) (MIC 31.3 μg/mL) alone and
62 previous studies reporting the antitubercular activity of β-sitos- in mixture with 4-hydroxybenzoic acid (12) (MIC 62.5 μg/mL).
63 terol, stigmasterol, and a (1:1) mixture in the MABA assay It should be mentioned that p-coumaric acid was previously
64 (Gutierrez-Lugo et al., 2005; Tan et al., 2008). It has been reported reported as inactive (MIC 4 128 μg/mL) in a study using the MABA
65 that the relatively polar “head groups” and flexible non-polar assay (Gutierrez-Lugo et al., 2005). The latter is a microplate-based
66 “phytyl tails” of sterols cause disruption in the mycobacterial cell colorimetric assay which provides a rapid high-throughput

Please cite this article as: Zhao, J., et al., Antitubercular activity of Arctium lappa and Tussilago farfara extracts and constituents. Journal
of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.034i
 4 J. Zhao et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Please cite this article as: Zhao, J., et al., Antitubercular activity of Arctium lappa and Tussilago farfara extracts and constituents. Journal
of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.06.034i