PHYTOTHERAPY RESEARCH Phytother. Res. 23, 99–103 (2009) Published onlineANTIBACTERIAL 11 August 2008 in Wiley InterScience IRIDOID GLUCOSIDES FROM EREMOSTACHYS LACINIATA (www.interscience.wiley.com) DOI: 10.1002/ptr.2568
99
Antibacterial Iridoid Glucosides from Eremostachys laciniata Masoud Modaressi1, Abbas Delazar1, Hossein Nazemiyeh1, Fatemeh Fathi-Azad1, Eileen Smith2, M. Mukhlesur Rahman2, Simon Gibbons2, Lutfun Nahar3 and Satyajit D. Sarker3* 1
School of Pharmacy, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Centre for Pharmacognosy and Phytochemistry, The School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX, UK 3 School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Co. Londonderry, Northern Ireland, UK 2
Eremostachys laciniata (L) Bunge (family: Lamiaceae alt. Labiatae; subfamily: Lamioideae) is one of the 15 endemic Iranian herbs of the genus Eremostachys. A decoction of the roots and flowers of E. laciniata has traditionally been taken orally for the treatment of allergies, headache and liver diseases. Three antibacterial iridoid glucosides, phloyoside I (1), phlomiol (2) and pulchelloside I (3) have been isolated from the rhizomes of this plant. The structures of these compounds were elucidated unequivocally by a series of 1D and 2D NMR analyses. The antibacterial activity and brine shrimp toxicity of these compounds were assessed using the resazurin microtitre assay and the brine shrimp lethality assay, respectively. All three iridoid glycosides 1–3 exhibited from low to moderate levels (MIC = 0.05–0.50 mg/mL) of antibacterial activity. Of these compounds, compound 3 was the most active, and displayed antibacterial activity against 9 of 12 different strains tested. The most noteworthy activity of 3 was against Bacillus cereus, penicillin-resistant Escherichia coli, Proteus mirabilis and Staphylococcus aureus with an MIC value of 0.05 mg/mL. Copyright © 2008 John Wiley & Sons, Ltd. Keywords: Eremostachys laciniata; Lamiaceae; iridoid; phloyoside I; pulchelloside I; phlomiol; antibacterial; chemotaxonomy.
INTRODUCTION
MATERIALS AND METHODS
Eremostachys laciniata (L) Bunge (family: Lamiaceae alt. Labiatae; subfamily: Lamioideae), a perennial herb with a thick root and pale purple or white flowers, is one of the 15 endemic Iranian species of the genus Eremostachys, and is also grown in other countries of the Middle-East Asia, Western Asia and Caucasus (GRIN Database, 2008; Mozaffrian, 1996). A decoction of the roots and flowers of E. laciniata has traditionally been taken orally for the treatment of allergies, headache and liver diseases (Said et al., 2002). A previous phytochemical study on E. laciniata revealed the presence of various mono- and sesquiterpenes in its essential oils (Navaei and Mirza, 2006). The crude extract of this plant was reported to possess a freeradical-scavenging property (Erdemoglu et al., 2006). As part of our on-going studies on the plants of the Iranian flora (Nazemiyeh et al., 2008, 2007; Delazar et al., 2007a, 2007b, 2006a, 2006b, 2006c, 2006d, 2005, 2004a, 2004b), we now report on the isolation, structure elucidation and bioactivity of three iridoid glucosides, phloyoside I (1), phlomiol (2) and pulchelloside (3) from the rhizomes of E. laciniata.
General experimental procedures. UV spectra were obtained in methanol using a Hewlett-Packard 8453 UV/vi spectrophotometer in MeOH. NMR spectra were recorded in CD3OD on a Bruker DRX 500 MHz NMR spectrometer (500 MHz for 1H and 125 MHz for 13C) using the residual solvent peaks as an internal standard. MS analyses were performed on a Finnigan MAT95 spectrometer. HMBC spectra were optimized for a long range JH-C of 9 Hz and a NOESY experiment was carried out with a mixing time of 0.8 s.
* Correspondence to: S. D. Sarkar, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Co. Londonderry, Northern Ireland, UK. E-mail:
[email protected] Copyright © 2008 John Wiley & Sons, Ltd. Copyright © 2008 John Wiley & Sons, Ltd.
Plant material. The rhizomes of Eremostachys laciniata (L) Bunge were collected during September–October 2005 from Ajabshir county in East Azarbaijan province in Iran (37° 36′ 46.7′′ N latitude, 46° 11′ 15.6′′ E longitude and altitude 1900 m above sea level). A voucher specimen (TUM-ADE 0204) has been retained in the herbarium of the Faculty of Pharmacy, Tabriz University of Medical Science, and in the herbarium of the Plant and Soil Science Department, University of Aberdeen, Scotland (ABD). Extraction and isolation of compounds. The dried and ground rhizomes of E. laciniata (100 g) were Soxhletextracted, successively, with n-hexane, dichloromethane and methanol (1.1 L each). The MeOH extract (2 g) was subjected to Sep-Pack fractionation using a step gradient of MeOH–water mixture (10:90, 20:80, 40:60, 60:40, 80:20 and 100:0). The preparative reversedphase HPLC analysis (Shim-Pak ODS column 10 μm, Received 12 February 2008 Phytother. Res. 23, 99–103 (2009) Revised 18 March 2008 DOI: 10.1002/ptr Accepted 20 March 2008
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Table 1. 1H NMR (500 MHz, coupling constant J in Hz in parentheses) and glucosides 1–3
13
C NMR (125 MHz in parentheses) data of iridoid
Chemical shift (δH) in ppm
Chemical shift (δC) in ppm
Position
1
2
3
1 3 4 5 6 7 8 9 10 11 11-OMe 1′ 2′ 3′ 4′ 5′ 6′
5.83 s 7.49 s – – 3.55 d (9.0) 3.82 d (9.0) – 2.47 s 1.02 s – 3.74 s 4.59 d (8.0) 3.19 dd (8.0, 9.0) 3.38 bt (9.0) 3.28* 3.32* 3.90 dd (2.0, 11.5) 3.66 dd (6.0, 11.5)
5.83 s 7.45 s – – 4.18 d (4.5) 3.66 d (4.5) – 2.51 s 1.39 s – 3.73 s 4.60 d (8.0) 3.20 bt (8.0) 3.38* 3.28* 3.29* 3.90 bd (12.0) 3.67*
5.67 d (2.0) 7.45 s – – 3.67* 3.43 dd (7.0, 10.0) 1.35 m 1.96 dd (2.0, 12.0) 1.37 d (6.5) – 3.73 s 4.58 d (8.0) 3.19 bt (8.0) 3.38* 3.28* 3.29* 3.90 dd (2.0, 11.5) 3.68*
Spectra obtained in CD3OD. All assignments were confirmed by
13
1
2
3
93.1 153.7 115.0 64.9 79.7 83.7 74.9 57.6 17.2 168.4 52.0 99.7 74.4 77.4 71.7 78.4 62.9
93.9 154.2 114.4 70.1 77.2 80.5 78.5 56.9 22.3 168.8 51.7 99.5 74.4 77.5 71.6 78.4 62.6
95.0 153.6 114.9 68.6 82.9 83.3 37.8 53.9 16.1 168.5 51.8 100.0 74.4 77.4 71.6 78.5 62.7
C DEPT135, COSY, NOESY, HMBC and HSQC experiments.
250 mm × 21.2 mm; mobile phase: 0–70 min gradient 4%–9% ACN in water; flow-rate: 20 mL/min, detection at 248 nm) of the 10% methanol Sep-Pack fraction afforded three iridoid glucosides, phloyoside I (1, 13.2 mg, tR = 12.3 min) (Kasai et al., 1994), phlomiol (2, 6.7 mg, tR = 13.6 min) (Zhang et al., 1991) and pulchelloside I (3, 6.7 mg, tR = 16.6 min) (Milz and Rimpler, 1978; El-Hela et al., 2000). All compounds 1–3 were identified unequivocally by a series of 1D and 2D NMR experiments, notably, 1H, 13C, 13C DEPT, 1H-1H COSY, 1H1 H NOESY, 1H-13C HMQC and 1H-13C HMBC (Table 1; Fig. 1) and HR-ESIMS analysis. The spectroscopic data of the known compounds were also compared with the respective published data. Phloyoside I (1). White amorphous solid; 13.2 mg; UV λmax (MeOH): 230 nm; HR-FABMS m/z 439.1451, C17H27O13 requires 439.1452; 1H NMR and 13C NMR (Table 1). Phlomiol (2). White amorphous solid; 6.7 mg; UV λmax (MeOH): 230 nm; HR-FABMS m/z 439.1451, C17H27O13 requires 439.1452; 1H NMR and 13C NMR (Table 1). Pulchelloside I (3). White amorphous solid; 6.7 mg; UV λmax (MeOH): 230 nm; HR-FABMS m/z 423.1503, C17H27O12 requires 423.1502; 1H NMR and 13C NMR (Table 1).
Antibacterial activity. Antibacterial activity of 1–3 was assessed against 12 strains of Gram-positive and Gramnegative bacteria including Bacillus cereus (NCTC 9689), Citrobacter freundii (NCTC 9750), Escherichia coli (NCIMB 8110), Escherichia coli (NCIMB 4174), Klebsiella aerogenes (NCTC 9528), Lactobacillus plantarum (NCIMB 6376), Micrococcus luteus (NCIMB 9278), Proteus mirabilis (NCIMB 60), Pseudomonas aeruginosa (NCTC 6750), Staphylococcus aureus (NCTC 10788), Staphylococcus aureus (MRSA) (NCTC 11940), Staphylococcus epidermidis (NCIMB 8558) using the 96-well microtitre-plate-based serial dilution method, incorporating resazurin as an indicator of cell growth (Sarker et al., 2007). Isosensitized nutrient broth was obtained from Oxoid, Basingstoke, Hampshire, England. Microtitre plates were from Serowel, Bibby sterilin, Stone, Staffs, UK. Eppendorf pipettes were purchased from Netheter-hinz-Gmbh, 22 331, Hamburg, Germany. Bacterial suspension (20 μL) in double strength nutrient broth at a concentration of 5 × 105 colony forming units (CFU)/mL was used. Test compounds (1–3) were dissolved in 10% aqueous DMSO to obtain a stock concentration of 1 mg/mL. Ciprofloxacin, a well-known broad-spectrum antibiotic, was used as a positive control. The minimum inhibitory concentration (MIC) was determined for each compound and compared with that of ciprofloxacin.
Figure 1. Structures of iridoid glycosides isolated from the rhizomes of Eremostachys laciniata.
Brine shrimp lethality assay. Shrimp eggs were purchased from The Pet Shop, Kittybrewster Shopping Complex, Aberdeen, UK. The bioassay was conducted following the procedure described by Meyer et al. (1982). The eggs were hatched in a conical flask containing 300 mL artificial seawater. The flasks were well aerated with the aid of an air pump, and kept in a water bath at 29– 30 °C. A bright light source was left on and the nauplii hatched within 48 h. The extracts were dissolved in 2% aq. DMSO to obtain a concentration of 1 mg/mL. These were serially diluted to obtain seven different
Copyright © 2008 John Wiley & Sons, Ltd.
Phytother. Res. 23, 99–103 (2009) DOI: 10.1002/ptr
ANTIBACTERIAL IRIDOID GLUCOSIDES FROM EREMOSTACHYS LACINIATA
concentrations. A solution of each concentration (1 mL) was transferred into clean sterile universal vials with a pipette, and aerated sea-water (9 mL) was added. About 10 nauplii were transferred into each vial with a pipette. A check count was performed and the number alive after 24 h was noted. LD50 values were determined using the Probit analysis method (Finney, 1971). Podophylotoxin, a well known cytotoxic lignan, was used as a positive control.
RESULTS AND DISCUSSION Reversed-phase preparative HPLC analysis of the methanol extract of the rhizomes of E. laciniata afforded three iridoid glucosides, which were identified unequivocally as phloyoside I (1), phlomiol (2) and pulchelloside I (3) on the basis of extensive 1D and 2D NMR data analyses. All three compounds (1–3) showed spectroscopic characteristics, especially, UV and NMR signals (Table 1), assignable to iridoid glucoside skeletons with a carbomethoxy group at C-4, and a methyl at C-8 (Boros and Stermitz, 1991). The HR-FABMS spectra of 1–3, pseudomolecular ions [M+1]+, respectively, at m/z 439.1451, 439.1451 and 423.1502, corresponding to the molecular formula, C17H26O13, C17H26O13 and C17H26O12, respectively. In the 1H and 13C NMR spectra of 1 (Table 1), in addition to the signals associated with a β-D-glucosyl and carbomethoxy moieties, there were signals corresponding to C-8 methyl (δH 1.02 and δC 17.2), olefinic methine (δH 7.49 and δC 153.7, C-3), three oxymethines at C-1 (δH 5.83 and δC 93.1), C-6 (δH 3.55 and δC 79.7) and C-7 (δH 3,82 and δC 83.7), a methine at C-9 (δH 2.47 and δC 57.6) and two oxygenated quarternary carbons at C-5 (δC 64.9) and C-8 (δC 74.9). While a 1H-1H COSY45 displayed all 1H-1H scalar couplings within the molecule, an HMBC together with an HSQC confirmed the 1H13 C connectivities, and thus the structure of the molecule. The relative stereochemistry at the chiral centres in 1, was established from the nOe interactions observed in the 1H-1H NOESY spectrum, especially, the strong nOe interactions between H-9 and H-7 established that both these protons were on the same face of the molecule. Thus, the identity of 1 was confirmed as phloyoside I, and the NMR data of 1 were comparable to the published data (Kasai et al., 1994). The 1H and 13C NMR data of 2 (Table 1) were similar to those of 1, with a few minor differences which originated from the differences in relative stereochemistry at the chiral centres of both molecules. Using the nOe interactions observed in the 1H-1H NOESY spectrum, the identity of 2 was confirmed as phlomiol. The NMR data of 2 were comparable to the published data (Zhang et al., 1991). In the 1H NMR spectrum of 3 (Table 1), all signals were similar to those observed in the case of compounds 1 and 2, with the exceptions that the C-8 methyl signal (δ 1.37) appeared as a doublet, and an additional methine signal at δ 1.35 as a multiplet was observed. In the 13C NMR spectrum (Table 1), the C-8 oxygenated quarternary signal was absent, and a methine signal (δ 37.8) was present instead. Finally, with the help of a 1H-1H COSY 45, 1H-13C HSQC, 1H-13C HMBC and 1 H-1H NOESY, the identity of compound 3 was conCopyright © 2008 John Wiley & Sons, Ltd.
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firmed as puchelloside I. The NMR data of 3 were comparable to the published data (Milz and Rimpler, 1978; El-Hela et al., 2000). To our knowledge, this is first report on the occurrence of iridoid glucosides 1–3 in the rhizomes of Eremostachys laciniata. However, Calis et al. (2007b), recently reported in a conference abstract, the presence of compounds 1 and 2 in the aerial parts of E. laciniata growing in Turkey. Iridoid glycosides are of common occurrence in the genus Phlomis (Delazar et al., 2004a; ISI Database, 2008; Combined Chemical Dictionary, 2008). Most recently, iridoid glycosides have also been reported from the genus Eremostachys (Delazar et al., 2004a, Calis et al., 2007a, 2007b), which is taxonomically close to Phlomis. Within the genus Phlomis, phloyoside I (1) was isolated previously from P. rotata and P. younghusbandii, and phlomiol (2) from P. tuberosa, P. longifolia and P. younghusbandii (ISI Database, 2008). However, pulchelloside I (3) was not previously reported either from the Phlomis or the Eremmostachys. Both the genera, Eremostachys and Phlomis, belong to the subtribe Lamieae of the family Lamiaceae (Azizian and Cutler, 1988; Delazar et al., 2004a), and they are morphologically similar. Anatomical and cytological studies on the species of these genera also established this close affinity between these two genera. During the preliminary chemotaxonomic studies on the family Lamiaceae using flavonoids as the markers, some degrees of similarities between these genera were also identified (Delazar et al., 2004a). Iridoids have been considered as valuable chemotaxonomic markers (Frederiksen et al., 1999), and in fact, they have been employed successfully to describe chemotaxonomic relationships among the taxa within various families, e.g. Acanthaceae, Bigoniaceae, Cornaceae, Oleaceae and Rubiaceae (Delazar et al., 2004a; ISI Database, 2008). Within the family Lamiaceae, iridoid glycosides have recently been employed as chemotaxonomic markers for the species of the genus Lamium (Alipieva et al., 2003). Therefore, the co-occurrence of iridoid glycosides, especially 1 and 2, in the closely related genera Eremostachys and Phlomis could be significant chemotaxonomically. All three iridoid glycosides 1–3 exhibited (Table 2) from low to moderate levels (MIC = 0.05–0.50 mg/mL) of antibacterial activity. Among these compounds, compound 3 was the most active, and displayed antibacterial activity against 9 of 12 different strains tested. The most noteworthy activity of 3 was against Bacillus cereus, penicillin-resistant Escherichia coli, Proteus mirabilis and Staphylococcus aureus with an MIC value of 0.05 mg/mL. None of the compounds (1–3) exhibited any inhibitory activities against Klebsiella aerogenes, Lactobacillus plantarum and methicillin-resistant Staphylococcus aureus. The antibacterial activity profiles of compounds 1 and 2, possibly owing to their structural similarities, were quite similar. The growth of Escherichia coli, penicillin resistant Escherichia coli, Micrococcus luteus and Staphylococcus epidermidis was inhibited only by compound 3. All compounds were active against Bacillus cereus, Citrobacter freundii, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. The brine shrimp lethality assay (BSL) has been used routinely in the primary screening of the crude extracts as well as the isolated compounds to assess the toxicity towards brine shrimps, which could also provide an Phytother. Res. 23, 99–103 (2009) DOI: 10.1002/ptr
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Table 2. Antibacterial properties of iridoid glucosides 1–3 MIC in mg/mL Bacterial strain
Bacillus cereus Citrobacter freundii Escherichia coli Escherichia coli (penicillin resistant) Klebsiella aerogenes Lactobacillus plantarum Micrococcus luteus Proteus mirabilis Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus aureus (MRSA) Staphylococcus epidermidis
1
2
3
0.50 0.50 – – – – – 0.50 0.50 0.25 – –
0.50 0.50 – – – – – 0.10 0.50 0.10 – –
0.05 0.50 0.25 0.05 – – 0.50 0.05 0.10 0.05 – 0.10
indication of possible cytotoxic properties of the test materials (Meyer et al., 1982). It has been established that the cytotoxic compounds usually show good activity in the BSL assay, and this assay can be recommended as a guide for the detection of antitumour and pesticidal compounds because of its simplicity and cost-effectiveness. In the BSL assay, none of these iridoid glycosides (1–3) showed any significant level of toxicity, and the LD50 values were >1.0 mg/mL,
Ciprofloxacin 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
× × × × × × × × × × × ×
10−8 10−7 10−7 10−6 10−6 10−7 10−7 10−8 10−8 10−8 10−8 10−7
compared with 2.80 μg/mL of the positive control, podophyllotoxin. Acknowledgement We thank the EPSRC National Mass Spectrometry Service Centre (Department of Chemistry, University of Wales Swansea, Swansea, Wales, UK) for MS analyses. NMR studies were performed in the School of Pharmacy, University of London, UK.
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