Bioactivity of two Turkish endemic Centaurea species, and their major constituents

Bioatividade e os principais constituintes químicos de duas espécies de Centaurea, endêmicas da Turquia

ARTIGO

Bioactivity of two Turkish endemic Centaurea species, and their major constituents

Bioatividade e os principais constituintes químicos de duas espécies de Centaurea, endêmicas da Turquia

Mohammad Shoeb^I; Stephen M. MacManus^II; Marcel Jaspars^III; Paul Kong-Thoo-Lin^IV; Lutfun Nahar^IV; Sezgin Celik^V; Satyajit D. SarkerVI, ^*

^IDepartment of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh

^IISchool of Pharmacy, The Robert Gordon University, Schoolhill, Aberdeen AB10 1FR, UK

^IIIMarine Natural Products Laboratory, Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, UK

^IVSchool of Life Sciences, The Robert Gordon University, St Andrew Street, Aberdeen AB25 1HG, UK

^VDepartment of Biology, Faculty of Science and Literature, Canakkale Onsekiz Mart University, 17020 Canakkale, Turkey

^VISchool of Biomedical Sciences, University of Ulster at Coleraine, Cromore Road, Coleraine BT52 1SA, Co. Londonderry, Northern Ireland, UK

ABSTRACT

The antioxidant activity, general toxicity and cytotoxicity of the methanol extracts of Centaurea urvillei subs. armata and C. mucronifera have been assessed, respectively, by the DPPH assay, the brine shrimp lethality and the MTT cytotoxicity assays. The reversed-phase HPLC analysis of the methanol extracts afforded two bioactive dibenzylbutyrolactone-type lignans, matairesinoside (1) and arctiin (2). The structures of these lignans were elucidated by comprehensive spectroscopic analyses as well as by the direct comparison of experimental data with respective literature data.

Keywords:Centaurea urvillei subs. armata, Centaurea mucronifera, Asteraceae, Compositae, lignan, arctiin, matairesinoside, DPPH, MTT, brine shrimp lethality assay.

RESUMO

A atividade antioxidante, a toxicidade geral e a citotoxidade dos extratos metanólicos de Centaurea urvillei subs. armata e C. mucronifera foram analisados, respectivamente, pelo ensaio DPPH e pelos ensaios de letalidade de Artemia salina e de citotoxidade MTT. A análise dos extratos metanólicos em CLAE de fase reversa apresentou duas lignanas biotivas do tipo dibenzilbutirolactona, matairesinosídeo (1) e arctiina (2). As estruturas destas lignanas foram elucidadas através de análises espectroscópicas completas bem como por comparação direta dos dados experimentais com os respectivos dados da literatura.

Unitermos:Centaurea urvillei subs. armata, Centaurea mucronifera, Asteraceae, Compositae, lignana, arctiina, matairesinosídeo, DPPH, MTT, Artemia salina, toxicidade.

INTRODUCTION

Centaurea urvillei subs. armata Wagenitz and C. mucronifera DC. (Family: Asteraceae alt. Compositae), are two Turkish endemic perennials that are distributed in the Mediterranean, the Middle and the Eastern Anatolian regions of Turkey (Wagenitz, 1975). To our knowledge, no reports on the isolation of any plant secondary metabolites or any pharmacological properties of these plants are available to date. However, many species of the genus Centaurea have long been used in traditional medicine to cure various ailments, e.g. diabetes, diarrhoea, rheumatism, malaria, hypertension, treatment of drug dependence etc., and a variety of secondary metabolites have been reported from different species of this genus (Sarker et al., 1997; Carlini et al., 2006). As a part of our continuing phytochemical and bioactivity studies on the species of the genus Centaurea (Shoeb et al., 2006a,b; 2005; 2004a,b; Sarker et al., 2005; 2001; 1998; 1997), we now report on the assessment of the extracts of C. urvillei subs. armata and C. mucronifera for antioxidant activity, general toxicity and cytotoxicity, respectively, by the DPPH assay, the brine shrimp lethality and the MTT cytotoxicity assays, and the isolation and identification of two major bioactive lignans, matairesinoside (1) and arctiin (2), from these plants.

MATERIAL AND METHODS

General

UV spectra were obtained in MeOH using a Hewlett-Packard 8453 UV-vis spectrometer. MS analyses were performed on a Quattro II triple quadrupole instrument. NMR spectra were recorded in CD₃OD on a Varian Unity INOVA 400 MHz NMR Spectrometer 400 (400 MHz for ¹H and 100 MHz for ¹³C) using the residual solvent peaks as internal standard. HPLC separation was performed using a Dionex prep-HPLC system coupled with Gynkotek GINA50 autosampler and Dionex UVD340S Photo-Diode-Array detector and/or A JASCO PU-1580 Intelligent HPLC Pump, coupled with JASCO DG-1580-53 Degasser and JASCO LG-1580-02 Ternary Gradient Unit. A Luna C₁₈ preparative (10 µM, 250 mm ´ 21.2 mm) and/or a Luna C₁₈ semi-preparative HPLC column (5 µM, 250 mm x 10 mm) were used. Sep-Pak Vac 35 cc (10 g) C₁₈ cartridge (Waters) was used for pre-HPLC fractions. HMBC spectra were optimised for a long range J_H-C of 9Hz and the NOESY experiment was carried out with a mixing time of 0.8s.

Plant material

The aerial parts of Centaurea urvillei subs. armata and C. mucronifera were collected from Anatolia, Turkey. Voucher specimens PHSH80012 and PHSH0013, respectively, for C. urvillei subs. armata and C. mucronifera have been retained in the herbarium of the Plant and Soil Science Department, University of Aberdeen, Scotland (ABD).

Extraction, isolation and structure elucidation

Dried ground aerial parts of C. urvillei subs. armata and C. mucronifera (100 g) were separately, Soxhlet-extracted, successively, with n-hexane, dichloromethane and methanol (MeOH) (1 L each). The MeOH extract (2.0 g) of C. urvillei was fractionated by solid-phase extraction method using a Sep-Pak C₁₈ (10 g) cartridge eluting with a step gradient: 40, 60, 80 and 100% MeOH in water (200 mL each). Prep-HPLC (eluted with a linear gradient- water:MeOH = 75:25 to 30:70 over 50 min followed by 70% MeOH for 10 min, 20 mL/min) of the Sep-Pak fraction, which was eluted with 40% MeOH, yielded compounds 1 (12.4 mg, t_R = 20.6 min) and 2 (56.0 mg, t_R = 24.5 min). The 'dereplication' of the Sep-Pak fractions of the MeOH extract (100 mg) of C. mucronifera using the HPLC-PDA analyses revealed the presence of exactly the same lignans (1 and 2) in the Sep-Pak fraction, which was eluted with 40% MeOH.

Matairesinoside (1): Gum, [a] ²³_D -48.8° (c 0.002, MeOH); UV l_max (MeOH): 279, 222; IR n_max (neat): 3373, 1760, 1600, 1514, 1452 and 1270 cm^-1; ESIMS m/z 543 [M+ Na]⁺; ¹H (400 MHz, CD₃OD) and ¹³C NMR (100 MHz, CD₃OD): as published data (Shoeb et al., 2004a).

Arctiin (2): Gum, [a] ²³_D -55.3° (c 0.0033, MeOH); UV l_max (MeOH): 279, 225; IR n_max (neat): 3459, 1765, 1591, 1460 and 1266 cm^-1; CIMS m/z 552 [M+ NH₄]⁺; ¹H (400 MHz, CD₃OD) and ¹³C NMR (100 MHz, CD₃OD): as published data (Shoeb et al., 2004a).

DPPH assay

2,2-Diphenyl-1-picrylhydrazyl (DPPH), molecular formula C₁₈H₁₂N₅O₆ , was obtained from Fluka Chemie AG, Bucks. Quercetin was obtained from Avocado Research Chemicals Ltd, Shore road, Heysham, Lancs. The method used by Takao et al. (1994) was adopted with suitable modifications (Kumarasamy et al. 2002). DPPH (4 mg) was dissolved in MeOH (50 mL) to obtain a concentration of 80 µg/mL.

Qualitative assay: Test samples (MeOH extract, 1 and 2) were applied on a TLC plate and sprayed with DPPH solution using an atomiser. It was allowed to develop for 30 min. The colour changes (purple on white) were noted.

Quantitative assay: The MeOH extract, and test compounds 1 and 2 were dissolved in MeOH to obtain a concentration of 1.0 mg/mL. Dilutions were made to obtain concentrations of 5 x 10^-1, 5 x 10^-2, 5 x 10^-3, 5 x 10^-4, 5 x 10^-5, 5 x 10^-6 5 x 10^-7, 5 x 10^-8, 5 x 10^-9, 5 x 10^-10 mg/mL. Diluted solutions (1.00 mL each) were mixed with DPPH (1.00 mL) and allowed to stand for 30 min for any reaction to occur. The UV absorbance was recorded at 517 nm. The experiment was performed in triplicate and the average absorption was noted for each concentration. The same procedure was followed for the positive standards (quercetin).

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 methanol extracts of two species and the test compounds (1 and 2) were dissolved in 20% aq. DMSO to obtain a concentration of 1 mg/mL. These were serially diluted two-times, and seven different concentrations were obtained. A solution of each concentration (1 mL) was transferred into clean sterile universal vials with pipette, and aerated sea-water (9 mL) was added. About 10 nauplii were transferred into each vial with pipette. A check count was performed and the number alive after 24 h was noted. LD₅₀s were determined using the Probit analysis method (Finney, 1971).

MTT cytotoxicity assay

CaCo-2 cells were maintained in Earle's minimum essential medium (Sigma), supplemented with 10% (v/v) foetal calf serum (Labtech Int.), 2 mM L-glutamine (Sigma), 1% (v/v) non-essential amino acids (Sigma), 100 IU/mL penicillin and 100 mg/mL streptomycine (Sigma). Exponentially growing cells were plated at 2 ´ 10⁴ cells cm^-2 into 96-well plates and incubated for 72 h before the addition of drugs. Stock solution of compounds was initially in DMSO or H₂O and further diluted with fresh complete medium.

The growth-inhibitory effects of the methanol extracts of two Centaurea species and the test compounds (1-2) were assessed using the standard tetrazolium MTT assay (Mosmann, 1983). After 72 h of incubation at 37 °C, the medium was removed, and 100 mL of MTT reagent (1 mg/mL) in serum free medium was added to each well. The plates were incubated at 37 °C for 4 h. At the end of the incubation period, the medium was removed and pure DMSO (200 mL) was added to each well. The metabolised MTT product dissolved in DMSO was quantified by reading the absorbance at 560 nm on a micro plate reader (Dynex Technologies, USA). The IC₅₀ values were calculated from the equation of the logarithmic line determined by fitting the best line (Microsoft Excel) to the curve formed from the data. The IC₅₀ value was obtained from the equation y = 50 (50% value).

RESULTS AND DISCUSSION

The MeOH extracts of C. urvillei subs. armata and C. mucronifera were tested for antioxidant activity, general toxicity and cytotoxicity, respectively, by the DPPH assay, the brine shrimp lethality and the MTT cytotoxicity assays. The crude MeOH extracts of C. urvillei subs. armata and C. mucronifera showed significant levels of free radical scavenging activity (antioxidant activity) (IC₅₀ = 51.6 x 10^-2 and 53.6 x 10^-2 mg/mL) in the DPPH assay (Table 1) which was due to the presence of two lignans, matairesinoside (1) and arctiin (2) with significant antioxidant properties (IC₅₀ = 2.2 x 10^-3 and 16.0 x 10^-2 and mg/mL, respectively).

The brine shrimp lethality assay has been demonstrated to be an effective, robust and rapid assay method for primary screening of compounds for potential cytotoxic activities (Meyer et al., 1982). The MeOH extracts of C. urvillei subs. armata and C. mucronifera were found to possess similar levels of general toxicities with the LD₅₀ values of 115.5 x 10^-2 and 120.0 x 10^-2 mg/mL, respectively (Table 1). Again, the lignans 1 and 2, as the major components present in the extracts, contributed to this activity (LD₅₀ values of 1.6 x 10^-2 and 9.8 x 10^-2 mg/mL, respectively).

While none of the extracts demonstrated any significant cytotoxicity against the CaCo-2 colon cancer cell line in the MTT assay (IC₅₀ = >1000 µg/mL), the isolated lignans (1 and 2) showed considerable cytotoxicity (Table 1). The IC₅₀ values for matairesinoside (1) and arctiin (2) were 220 and 288 µM. It has previously been observed that it is not at all surprising to have active compounds from inactive extracts or fractions as the amounts of active compounds present in the amounts of extracts or fractions tested can be too small to show any activity (Shoeb et al., 2006a).

Reversed phase HPLC analyses of the 40% Sep-Pak fraction of the methanol extracts of C. urvillei subs. armata and C. mucronifera afforded two known bioactive dibenzylbutyrolactone-type lignans, matairesinoside (1) and arctiin (2). The structures of these compounds were elucidated by comprehensive spectroscopic analyses, and also by direct comparison with the respective published data (Shoeb et al., 2004a). Compounds 1 and 2 displayed characteristic UV absorption maxima of dibenzylbutyrolactone-type lignans (Shoeb et al., 2004a). The ¹H and ¹³C NMR spectra of these compounds also supported this fact. A CIMS spectrum of 2 revealed the [M+NH₄]⁺ ion peak at m/z 552, suggesting Mr = 534, and the molecular formula C₂₇H₃₄O₁₁. The ¹H and ¹³C NMR spectral data of 2 were identical to those published for arctiin (2) (Shoeb et al., 2004a). A combination of HMQC, HMBC, COSY and NOESY 2D NMR spectral analyses led to the unambiguous assignment of all ¹H and ¹³C NMR signals of 2 and confirmed unequivocally its identity as arctiin (2).

The ¹H and ¹³C NMR data of 1 were similar to those of arctiin (2) with the exception that one methoxyl signal was missing. The CIMS spectrum of 1 revealed [M+NH₄]⁺ ion at m/z 538, suggesting Mr = 520, and the molecular formula C₂₆H₃₂O₁₁ which also confirmed the findings from the NMR data that it contained 14 mass units less than arctiin (2), i.e. instead of a methoxyl group, it had a hydroxyl group present. The ¹H and ¹³C NMR data of 1 were in good agreement with the published data of matairesinoside (Shoeb et al., 2004a). Using the HPLC-PDA-based 'dereplication' protocol, the lignans 1 and 2 were also unambiguously identified in the 40% Sep-Pak fraction of the MeOH extract of C. mucronifera, and so was not processed for further purification.

These lignans were previously reported from a few other species of the genus Centaurea (ISI database, 2006; Shoeb et al., 2004a). However, this is the first report on the occurrence of matairesinoside (1) and arctiin (2) in C. urvillei subs. armata and C. mucronifera. The co-occurrence of these dibenzylbutyrolactone lignans (1 and 2) within the genus Centaurea might be chemotaxonomically significant.

The overall bioactivity profiles of the MeOH extracts of C. urvillei subs. armata and C. mucronifera were almost identical (Table 1). It is interesting, but not unexpected, to note that both extracts have the same chemical profiles as well, and contain matairesinoside (1) and arctiin (2) as the major bioactive compounds.

ACKNOWLEDGEMENTS

We thank the EPSRC National Mass Spectrometry Service Centre (Department of Chemistry, University of Wales Swansea, Swansea, Wales, UK) for CIMS analyses.

Received 01/19/07. Accepted 04/20/07

• Carlini EA, Rodrigues E, Mendes FR, Tabach R, Gianfratti B 2006. Treatment of drug dependence with Brazilian herbal medicines. Rev Bras Farmacogn 16: 690-695.
• Finney D 1971. Probit analysis, 3^rd eD., Cambridge University Press, Cambridge.
• ISI Database 2006. Institute for Scientific Information, UK Available on-line through Web of Science at: http://wos.mimas.ac.uk
• Kumarasamy Y, Fergusson M, Nahar L, Sarker SD 2002. Biological activity of moschamindole from Centaurea moschata Pharm Biol 40: 307-310.
• Meyer BN, Ferrigni RN, Putnam JE, Jacobsen LB, Nichols DE, Mclaughlin JL 1982. Brine shrimp - A convenient general bioassay for active plants constituents. Planta Med 45: 31-34.
• Mosmann T 1983. Rapid colorimetric assay for the cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63.
• Sarker SD, Kumarasamy Y, Shoeb M, Celik S, Yucel E, Middleton M, Nahar L 2005. Antibacterial and antioxidant activities of three Turkish species of the gemus Centaurea Oriental Pharmacy and Experimental Medicine 5: 246-250.
• Sarker SD, Laird A, Nahar L, Kumarsamy Y, Jaspars M 2001. Indole alkaloids from the seeds of Centaurea cyanus (Asteraceae). Phytochemistry 57: 1273-1276.
• Sarker SD, Dinan L, Sik V, Underwood E, Waterman PG 1998. Moschamide: an unusual alkaloid from the seeds of Centaurea moschata. Tetrahedron Lett 39: 1421-1424.
• Sarker SD, Savchenko T, Whiting P, Sik V, Dinan LN 1997. Moschamine, cis-moschamine, moschamine, moschamindole and moschamindolol: four novel indole alkaloids from Centaurea moschata Nat Prod Lett 9: 189-199.
• Shoeb M, MacManus SM, Kumarasamy Y, Jaspars M, Nahar L, Kong-Thoo-Lin P, Nazemiyeh H, Sarker SD 2006a. Americanin, a bioactive dibenzylbutyrolactone lignan, from the seeds of Centaurea americana Phytochemistry 67: 2370-2375.
• Shoeb M, MacManus SM, Jaspars M, Trevidu J, Nahar L, Kong-Thoo-Lin P, Sarker SD 2006b. Montamine, a unique dimeric indole alkaloid, from the seeds of Centaurea montana (Asteraceae), and its in vitro cytotoxic activity against the CaCo-2 colon cancer cells. Tetrahedron 62: 11172-11177.
• Shoeb M, Celik S, Jaspars M, Kumarasamy Y, MacManus SM, Nahar L, Kong-Thoo-Lin P, Sarker SD 2005. Isolation, structure elucidation and bioactivity of schischkiniin, a unique indole alkaloid from the seeds of Centaurea schischkini Tetrahedron 61: 9001-9006.
• Shoeb M, Rahman MM, Nahar L, Jaspars M, MacManus S, Delazar A, Sarker SD 2004a. Bioactive lignans from the seeds of Centaurea macrocephala. DARU - Journal of Tehran School of Pharmacy 12: 87-93.
• Shoeb M, Jaspars M, MacManus SM, Majinda RRT, Sarker SD 2004b. Epoxylignans from the seeds of Centaurea cyanus Biochem Syst Ecol 32: 1201-1204.
• Takao T, Watanabe N, Yagi I, Sakata K 1994. A simple screening method for antioxidants and isolation of several antioxidants produced by marine bacteria from fish and shellfish. Biosci Biotech Biochem 58: 1780-1783.
• Wagentiz G 1975. Flora of Turkey and the East Aegean Islands, Davis, P.H, Eds., University Press, Edinburgh, 5.

Publication Dates

• Publication in this collection
  20 July 2007
• Date of issue
  June 2007

History

• Accepted
  20 Apr 2007
• Received
  19 Jan 2007