Bioactive Constituents of Conyza albida

• Abstract
• Introduction
• Material and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Isolation and purification of compounds 1 - 4
• Biological evaluation
• Results and Discussion
• Acknowledgements
• References

Abstract

The bioactivity-guided fractionation of an active chloroform extract of Conyza albida led to the isolation of three alkenynes, deca-4,6-diyn-2-(Z)-enoic methyl ester (1), deca-4,6-diyn-2-(Z)-enoic ethyl ester (2) and deca-2,4-diene-4-hydroxy-6-yn-1,4-olide (3), and the terpenoid spathulenol (4), as the active toxic metabolites in the Artemia sp. lethality test. When tested in the KB cell cytotoxicity assay, compounds 1 - 4 demonstrated IC₅₀ values of 52.2, 38.4, 117.9, and 83.8 μM, respectively. All compounds studied were inactive in the DNA methyl green and DNA strand scission assays, while compounds 3 and 4 showed moderate activity as inhibitors of human topoisomerase I. Compound 2 is reported here for the first time.

Introduction

Conyza albida Willd. ex Sprengel (Compositae) is a native species of Argentina and other parts of South America and introduced to Europe. Formerly, it was included as a synonym of C. bonariensis var. microcephala Cabr., but there is enough evidence to consider it a valid entity at the species level [1]. Conyza albida is reported to have use as for its expectorant, antitussive, and anti-inflammatory activities [2]. Since C. albida usually grows together with C. bonariensis populations, it is believed that both species are useful in the treatment of urinary affections, liver diseases, stomach ulcers, and to wash sores as well as antihelmintics, digestives and diuretics [3].

Despite the fact that many Conyza species have ethnomedical uses, only one study has been published on the cytotoxic activity of their extracts, which reported that C. filaginoides methanol extract showed weak activity against colon adenocarcinoma (HT 29) cells (ED₅₀ = 31 μg/ml) [4]. It is important to note that C. dioscoridis, also reported in the literature as having cytotoxic activity [5], is no longer a valid name for the species since nowadays it is considered as Pluchea dioscoridis (L.) D.C. [6]. To our knowledge, there have been no previous phytochemical studies performed on the constituents of C. albida. We present herein the results on the bioactivity-guided fractionation of an active extract of the leaves of C. albida and the evaluation of the activity of the pure compounds against Artemia sp., KB cells, and inhibition against human topoisomerase I. Activity in the DNA methyl green and DNA strand-scission assays was also examined.

Material and Methods

General experimental procedures

The UV and IR spectra were recorded on a Shimadzu UV 260 and a Nicolet 5-SXC-FTIR spectrophotometer, respectively. The optical rotation values were obtained on a JASCO P1020 spectropolarimeter. ¹H- and ¹³C-NMR spectra were recorded on a Bruker AC-200 NMR spectrometer at 200.13 and 50.32 MHz, respectively, using CDCl₃ as solvent and TMS as an internal standard. Chemical shifts are given in ppm downfield from TMS and coupling constants are measured in Hz. NOESY experiments were obtained using standard Bruker software. Gas-chromatography-mass spectrometry (GC-MS) was performed on a Perkin-Elmer Q-MASS 910 instrument. Chromatographic separations were achieved by vacuum-liquid chromatography (VLC) and column chromatography (CC) using silica gel 60 (40 - 63 μm), or by centrifugal chromatography in a Chromatotron Model 7924T using silica gel 60 PF₂₅₄ (Merck 7749) plates (2 mm thick, total silica length 7 cm). All solvents were distilled before use; preparative TLC was performed on silica gel 60 G F₂₅₄, 16 × 5 cm (L × H) plates, 0.2 mm thick, 15 mg maximum sample loading, by multiple development using hexane/acetone in different proportions. Analytical TLC was performed in the same manner and inspected under UV light (254 nm) or using vanillin/H₂SO₄ as detection reagent.

Plant material

The leaves of Conyza albida were collected in Córdoba, Argentina, on March 1995. The plant was identified by one of us (LAE), and a voucher specimen (Silva s.n., CORD 301) has been deposited at the Museo Botánico Córdoba, Córdoba, Argentina.

Extraction and isolation

Dried ground leaves of Conyza albida (1 kg) were extracted with CHCl₃ by standing overnight at room temperature (3 × 3 l). The extract was evaporated to dryness at 40 °C to give a gummy residue (113 g) that was suspended in MeOH/H₂O (3 : 1) and partitioned between hexane (4 × 3 l). The methanolic phase was evaporated at reduced pressure (40 °C) and partitioned successively between Et₂O (3 × 300 ml), CH₂Cl₂ (2 × 300 ml) and EtOAc (2 × 200 ml). All the extracts, including the water extract, were concentrated to dryness and tested in the brine shrimp toxicity test (BSTT); the hexane and Et₂O extracts gave positive results with LC₅₀ = 99 μg/ml and LC₅₀ = 96 μg/ml, respectively.

Isolation and purification of compounds 1 - 4

The ethyl ether extract (6.2 g) was fractionated by VLC using a Büchner type funnel with fibrous glass frit (disc diam. 150 mm, capacity 400 ml) filled with 270 g silica gel 60; each fraction (250 ml) was eluted with fr. 1, hexane (1 vol.) and hexane/acetone 4 : 1 (1 vol.), these two volumes were combined; fr. 2, hexane/acetone 4 : 1; fr. 3, 4, hexane/acetone 7 : 3; fr. 5, 6, hexane/acetone 3 : 2; fr. 7, 8, hexane/acetone 1 : 1; fr. 9, hexane/acetone 2 : 3; fr. 10, acetone and fr. 11, acetone/methanol 4 : 1. Fraction 2 (667.0 mg) was purified by column chromatography (CC) on silica gel 60 (30 g) (2 × 18 cm), eluting with hexane/acetone 4 : 1; a total of 20 fractions (15 ml each) were collected. Subfraction 2 - 3 (42.3 mg) showed an active spot in the UV light; purification by centrifugal chromatography, hexane/acetone 8 : 2, yielded pure compounds 1 (9.8 mg) [7], [8] and 2 (9.2 mg), R_f 0.37 and 0.46 (hexane/acetone 9 : 1), respectively. Subfraction 2 - 12 (26 mg) purified by preparative TLC, hexane/acetone 9 : 1 (3 developments), afforded compounds 1 (4.4 mg) and 4 (3.5 mg) [9]. Sesquiterpene 4 showed by TLC R_f 0.29 (hexane/aetone 9 : 1), and [α]²⁵ _D = + 9.8 (c = 0.1, CHCl₃). Subfraction 2 - 18, purified in the same manner, gave compound 3 (8 mg), R_f 0.32 (hexane/acetone 8 : 2) [7], [8]. The hexane extract (18.3 g) was fractionated by VLC eluting with hexane (500 ml), hexane/acetone 9 : 1 (4 × 500 ml), and acetone (500 ml). Subfraction 3 (2.2 g) was chromatographed by CC (silica gel 60, 90 g) eluting with hexane (250 ml) and hexane/acetone 9 : 1 (1000 ml); the fractions (20 ml each) were monitored by TLC and those containing the UV-active components were pooled and purified in the same fashion as above using hexane/acetone 99 : 1; 49 : 1, 95 : 5; 93 : 7 and pure acetone; subfraction 3 - 12 (21 mg), after preparative TLC, hexane/acetone 9 : 1 (developed twice), gave compound 1, which was not obtained in purified form. Compounds 1 - 3 were air and light sensitive and were accordingly protected in a sealed amber glass ampoule under a nitrogen atmosphere after purification.

Deca-4,6-diyn-2-(Z)-enoic ethyl ester (2): colorless oil; UV (λ_max, MeOH): 307, 290, 225 and 216 nm; IR (AgCl): ν = 2969, 2849, 2223, 1727, 1707, 1221 cm^-1; EIMS: m/z = 190 (11), 161 (4), 145 (9), 133 (22), 120 (12), 115 (10), 105 (16), 91 (21), 79 (16), 77 (20), 62 (31), 51 (66), 39 (100); ¹H NMR (CDCl₃): δ = 6.20 (1H, d, J = 11.2 Hz, H-3), 6.13 (1H, d, J = 11.2 Hz, H-2), 4.24 (2H, q, J = 7.2 Hz, H-1′), 2.35 (2H, t, J = 7.1 Hz, H-8), 1.58 (2H, m, H-9), 1.32 (3H, t, J = 7.2 Hz, H-2′), 1.00 (3H, t, J = 7.2 Hz, H-10); ¹³C NMR (CDCl₃): δ = 164.4 (C-1), 122.2 (C-2), 131.3 (C-3), 71.0 (C-4), 89.9 (C-5), 65.3 (C-6), 86.4 (C-7), 21.6 (C-8), 21.8 (C-9), 13.5 (C-10), 60.6 (C-1′), 14.1 (C-2′).

Biological evaluation

Brine shrimp toxicity test (BSTT): The BSTT was performed according to standard protocols [10]. The LC₅₀ values were determined for the chloroform extract and different fractions in μg/ml, and for the isolated compounds in μM, using the Finney probit analysis computer program [11]. Berberine chloride (Sigma) was used as positive control (LC₅₀ = 22.0 μM).

KB cell cytotoxicity assay: KB cell cytotoxicity was determined as described in [10] using the human oral epidermal carcinoma cellular line (KB) provided by the University of Illinois at Chicago, Chicago, IL, USA. Three concentrations were tested (in triplicate), and two independent experiments were run. Results were expressed as the dose that inhibited 50 % control growth (IC₅₀). The values were estimated from a semi-log plot of the drug concentration against the percentage of viable cells. Colchicine (Sigma) was used as a positive control (IC₅₀ = 0.05 μM).

DNA methyl green bioassay (DNA-MG): Interaction with DNA was determined using 96-well microplates [10] employing the DNA methyl green reagent (Sigma). The decrease in the initial absorbance of each sample was read at 655 nm using a Biorad Microplate Model 450 Reader (Biorad, New York, NY, USA). Doxorubicin hydrochloride was used as a positive control (IC₅₀ = 52 μM).

DNA strand-scission assay: The DNA strand-scission assay was adapted from a previously described procedure [12]. Briefly, samples were dissolved in DMSO and assayed at 100, 10 and 1 μg/ml. The assay reaction mix contained 25 mM cacodylate buffer pH 7.0, 0.3 mM CuCl₂ and 250 ng covalently closed circular pBR322 (supercoiled) as a substrate. The assay was initiated by the addition of the sample, incubated for 30 min at room temperature while protected from the light, and terminated by addition of stop reagents. The reaction mixture was analyzed by electrophoresis at 40 volts for 4 h on a 1 % agarose gel.

Inhibition of topoisomerase I activity: Inhibition of topoisomerase I activity was determined using a relaxation assay previously described [10], using purified human topoisomerase I (Topogen, Columbus, Ohio, USA) and Plasmid pHOT1 (Topogen) as a substrate. The compounds were pre-dissolved in DMSO and tested at 100, 10 and 1 μg/ml in 10 % DMSO (v/v). The products of the reaction, previously extracted with chloroform-isoamyl alcohol, were submitted to a 1 % agarose gel electrophoresis procedure for 4 h at 36 V at room temperature. Negatives of the gel pictures were scanned with a Hewlett Packard ScanJet 11p interfaced with a computer in order to quantify enzyme inhibition. The bands were analyzed with a Jandel SigmaScan image measurement analyzing program.

Results and Discussion

The total chloroform extract was suspended in MeOH-H₂O (20 %) and partitioned between several solvents. The hexane and diethyl ether extracts concentrated the lethal activity against Artemia sp. (BSTT) [10], which was used as a monitor, with LC₅₀ = 96 and 99 μg/ml, respectively. The diethyl ether extract was fractionated and the fractions were tested in the BSTT; the activity was mostly concentrated in the less polar fractions. The most active fraction was analyzed by GC-MS showing three major peaks with molecular ions of m/z 176 (1), 190 (2), and 162 (3) suggesting a high degree of unsaturation in each compound. Further work up on this fraction afforded pure compounds 1 to 4 as the active constituents. The degradation patterns of compounds 1 and 2 were almost superimposable, but different by 14 amu, while compound 3 displayed differences from 1 and 2 only in the higher mass ions. Compounds 1 and 2 showed in their UV spectra the typical diyne chromophore absorption at 307, 290, 225 and 216 nm. The IR spectra for both compounds displayed absorptions for the acetylene bonds near 2220 cm^-1 and a carbonyl signal near 1730 cm^-1. The ¹H-NMR spectra showed similar profiles with signals for an n-propyl residue and for two vinyl protons, together with signals for a methoxy group and an ethoxy group for 1 and 2, respectively. Further analysis on these compounds led to the identification of deca-4,6-diyn-2-(Z)-enoic methyl ester or trans-lachnophyllum methyl ester (1) and deca-4,6-diyn-2-(Z)-enoic ethyl ester or lachnophyllum ethyl ester (2). The stereochemistry of the double bond was established to be Z due to the coupling constant value of the vinyl protons (J = 11.2 Hz).[]

While alkenyne 2 is reported here for the first time, its trans isomer is already known as a synthetic product [13]. Compound 3 revealed by IR the absorption of an acetylenic bond at 2213 cm^-1 and of a carbonyl group at 1752 cm^-1 suggesting the presence of an α,β-unsaturated γ-lactone ring with a free α-position (an additional band at 1782 cm^-1 was observed), while in the ¹H-NMR spectrum it showed signals for three vinyl protons; the compound was finally identified as deca-2,4-diene-4-hydroxy-6-yn-1,4-olide or trans-lachnophyllum lactone (3). The stereochemistry of the C-4, C-5 double bond was confirmed by the cross-correlation peak seen between H-3 and H-5 in the NOESY experiment. Compounds 1 and 2 accounted also for the observed activity of the hexane extract. Compounds 1 and 3 were already known to exist in Conyza species and were previously isolated from C. bipinnata [7] and C. bonariensis [8]. Compound 4 was identified as the sesquiterpene (+)-spathulenol by comparison with published spectral data [9]. (+)-Spathulenol (4) is a common component of essential oils of many genera, but in Conyza species was reported only once from the essential oil of C. canadensis [14].

Alkenynes 1 and 2 showed potent activity in the BSTT (LC₅₀ = 6.8 μM and 6.8 μM, respectively) and were moderately active against KB cell line (IC₅₀ = 52.3 μM and 38.4 μM, respectively). Compound 3 showed an LC₅₀ = 32.1 μM in the BSTT and was less active [15] in the KB cell cytotoxicity assay (IC₅₀ = 117.9 μM) (Table [1]).

The sesquiterpene (+)-spathulenol (4) also demonstrated toxicity in BSTT (LC₅₀ = 19.4 μM) and was moderately cytotoxic to KB cells (IC₅₀ 83.8 μM); the latter activity has been tested previously with similar results [16] (Table [1]).

Further mechanistic assays were performed for the pure compounds 1 to 4 in the DNA methyl green [10], the DNA strand-scission [12] and in the topoisomerase I inhibition assays [10]. Then, in order to determine if cytotoxicity to KB cells was due to the capacity of the compounds to interact with DNA, a colorimetric microassay was carried out. This method determines the ability of a compound to interact with the genetic material through the displacement of methyl green from DNA, which leads to the decoloration of the MG-DNA complex [17]. No interaction of the compounds 1 - 4 could be observed at 100 μg/ml final concentration.

Because of the importance in suggesting new mechanistic principles that can be used to mediate DNA cleavage, we investigated the DNA strand-scission activity of the isolated compounds. This mechanism occurs when the compound interacts with DNA in the presence of important cofactors like Fe²⁺ or Cu²⁺, which in presence of O₂ acts like an enzyme and mediates the breakage and release of DNA bases [18]. DNA cleavage assays employ supercoiled covalently closed circular DNA as a substrate. A single cleavage event within any of the several thousand base pairs will result in relaxation of the supercoiled DNA form to relaxed DNA, the latter of which is separable by agarose gel electrophoresis.

Inhibition of human DNA topoisomerase is a major mechanism through which chemotherapeutic agents exert direct cytotoxicity to human cells [19]. No inhibition in the activity of the type I enzyme using agarose gel electrophoresis was detected in the presence of compounds 1 and 2. However, topoisomerase I-dependent DNA relaxation was prevented by exposure to compounds 3 and 4 at 100 μg/ml (Figs. [1], [2]). Drug-induced inhibition of topoisomerase I-mediated DNA relaxation was measured. Compound 3 showed a 21 % of inhibition while compound 4 showed 39 % inhibition. These results may partially justify the cytotoxicity observed for these compounds. It is interesting to note that compound 4 exerts selective cytotoxicity to the SKMel-2 cellular line (human melanoma cells) and this could be a possible mechanism of action [16]. Finally, polyenes and polyacetylenes are known to display several biological activities [20], [21] and this may be responsible for the activity displayed by Conyza extracts and could justify their use in ethnomedicine.

Acknowledgements

This research was supported by Fundación Antorchas, CONICOR, UBACyT and SeCyT.

References

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  PubMed

Dr. Gloria L. Silva

Departamento de QuÕmica OrgÄnica

Facultad de Ciencias Químicas U.N.C.

IMBIV-CONICET, Pabellón de Ciencias II

Ciudad Universitaria

5000 Córdoba

Argentina

Email: silvagl@dqo.fcq.unc.edu.ar

Fax: 54-351-4333030

References

• 1 Ariza Espinar L. Conyza albida (Compositae) en Argentina.  Boletín de la Sociedad Argentina de Botánica. 1982;  21 269-71
  PubMedSearch in Google Scholar
• 2 Marzocca A. Conyza bonariensis. Vademécum de Malezas Medicinales de la Argentina, Indígenas y Exóticas. Editorial Hemisferio Sur Buenos Aires, Argentina; 1997: 259
• 3 González A, Ferreira F, Vázquez A, Moyna P, Alonso Paz E. Biological screening of Uruguayan medicinal plants.  Journal of Ethnopharmacology. 1993;  39 217-20
  CrossrefPubMedSearch in Google Scholar
• 4 Gutierrez-Lugo M T, Barrientos-Benitez T, Luna B, Ramirez-Gamma R M, Bye R, Linares E, Mata R. Antimicrobial and cytotoxic activities of some crude drug extracts from Mexican medicinal plants.  Phytomedicine. 1996;  2 341-7
  PubMedSearch in Google Scholar
• 5 Al-Yahya M A, Mossa J S, Al-Meshal I A, Antoun M D, Mc Cloud T G, Cassady J M, Jacobsen L B, McLaughlin J L. Phytochemical and biological studies on Saudi medicinal plants part 9. Antitumor testing.  International Journal of Crude Drug Research. 1985;  23 45-66
  PubMedSearch in Google Scholar
• 6 Anderberg A A. Taxonomy and phylogeny of the tribe Plucheeae (Asteraceae).  Plant Systematic Evolution. 1991;  176 145-77
  PubMedSearch in Google Scholar
• 7 Bohlmann F, Jakupovic J. 8-Oxo-α-selinen und neue Scopoletin-derivate aus Conyza-arten.  Phytochemistry. 1979;  18 1367-70
  CrossrefPubMedSearch in Google Scholar
• 8 Sanz J F, Marco J A. Ein neues Butenolid aus Conyza bonariensis .  Liebigs Annalen Chemie. 1991;  399-400
  PubMedSearch in Google Scholar
• 9 Krebs H C, Rakotoarimanga J V, Habermehl G G. Isolation of spathulenol and (-)-caryophyllene oxide from Vernonia mollissima Don and ¹H and ¹³C reassignment by two-dimensional NMR spectroscopy.  Magnetic Resonance in Chemistry. 1990;  28 124-8
  CrossrefPubMedSearch in Google Scholar
• 10 Mongelli E, Romano A, Desmarchelier C, Coussio J, Ciccia G. Cytotoxic 4-nerolidylcatechol from Pothomorphe peltata inhibits topoisomerase I activity.  Planta Medica. 1999;  65 376-8
  PubMedSearch in Google Scholar
• 11 McLaughlin J. Methods in Plant Biochemistry. Crown gall tumors on potato discs and brine shrimp lethality: two bioassays for higher plant screening and fractionation. In: Harborne JB, editor . Academic Press New York; 1991: 1-32
• 12 Sugiyama H, Ehrenfeld G N, Shipley J B, Kilkuskie R E, Chang L H, Hecht S M. DNA strand scission by bleomycin group antibiotics.  Journal of Natural Products. 1985;  48 869-77
  PubMedSearch in Google Scholar
• 13 Carpita A, Neri D, Rossi R. Stereocontrolled synthesis of naturally-occurring poliacetylenes characterized by (E)-1-en-3-yne, (E)-1-en-3,5-diyne, (1E,5E)-1,5-dien-3-yne, and (1E,7E)-1,7-dien-3,5-diyne moieties.  Gazzetta Chimica Italiana. 1987;  117 481-97
  PubMedSearch in Google Scholar
• 14 Miyazawa M, Yamamoto K, Kameoka H. The essential oil of Erigeron canadensis .  Journal of Essential Oil Research. 1992;  4 227-30
  PubMedSearch in Google Scholar
• 15 Köning G, Wright A, Sticher O, Angerhofer C, Pezzuto J. Biological activities of selected marine natural products.  Planta Medica. 1994;  60 532-7
  PubMedSearch in Google Scholar
• 16 Fullas F, Hussain R A, Chai H B, Pezzuto J M, Soejarto D D, Kinghorn A D. Cytotoic constituents of Baccharis gaudichaudiana .  Journal of Natural Products. 1994;  57 801-7
  PubMedSearch in Google Scholar
• 17 Bonjean K, De Pauw-Gillet M, Bassleer R, Quentin-Leclercq J, Angenot L, Wright A. Cytotoxic activities of Colombian plant extracts on Chinense hamster lung fibroblasts.  Phytotherapy Research. 1996;  10 159-60
  PubMedSearch in Google Scholar
• 18 Suffness M, Pezzuto J. Methods in Plant Biochemistry. Assays related to cancer drug discovery. In: Harborne JB, editor Academic Press New York; 1991: 71-133
• 19 Liu L F. DNA Topoisomerase poisons as antitumor drugs.  Annual Review of Biochemistry. 1989;  58 351-75
  PubMedSearch in Google Scholar
• 20 Jung J H, Kim Y, Lee C O, Kang S S, Park J H, Im K S. Cytotoxic constituents of Saussurea lappa .  Archives of Pharmaceutical Research. 1998;  21 153-56
  PubMedSearch in Google Scholar
• 21 Takaishi Y, Okuyama T, Masuda A, Nakano K, Murakami K, Tomimatsu T. Acetylenes from Cirsium japonicum .  Phytochemistry. 1990;. 29 3849-52
  PubMed

Dr. Gloria L. Silva

Departamento de QuÕmica OrgÄnica

Facultad de Ciencias Químicas U.N.C.

IMBIV-CONICET, Pabellón de Ciencias II

Ciudad Universitaria

5000 Córdoba

Argentina

Email: silvagl@dqo.fcq.unc.edu.ar

Fax: 54-351-4333030