Original Papers
Antiproliferative Activity of Artemisia asiatica Extract and Its Constituents on Human Tumor Cell Lines
Authors
Zsuzsanna Hajdú 1, Judit Hohmann 1, Peter Forgo 1, Imre Máthé 1, 2, Judit Molnár 3, István Zupkó 3
Affiliations
1 2 3
Key words " Artemisia asiatica l " Asteraceae l " antiproliferative activity l " sesquiterpene lactones l " monoterpenes l " flavonoids l
received revised accepted
May 19, 2014 Sept. 9, 2014 Sept. 15, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383146 Published online October 8, 2014 Planta Med 2014; 80: 1692–1697 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Judit Hohmann University of Szeged Department of Pharmacognosy Eotvos u. 6 6720 Szeged Hungary Phone: + 36 62 54 64 53 Fax: + 36 62 54 57 04 [email protected]
Institute of Pharmacognosy, University of Szeged, Szeged, Hungary Institute of Ecology and Botany, Centre for Ecological Research, Hungarian Academy of Sciences, Vácrátót, Hungary Institute of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged, Hungary
Abstract !
The extract of Artemisia asiatica herb with antiproliferative activity against four human tumor cell lines (A2780, A431, HeLa, and MCF7) was analyzed by the MTT assay, and bioassay-directed fractionation was carried out in order to identify the compounds responsible for the cytotoxic activity. Guaianolide (1–4), seco-guianolide (5), germacranolide (6) and eudesmanolide sesquiterpenes (7), monoterpenes (8, 9), including the new compound artemisia alcohol glucoside (8), and flavonoids (10–16) were isolated as a result of a multistep chromatographic procedure (CC, CPC, PLC, and gel filtration). The compounds were identified by means of UV, MS, and NMR spectroscopy, including 1H‑and 13C‑NMR, 1H-1H COSY, NOESY, HSQC, and HMBC experiments. The isolated compounds 1–16 were evaluated for their tumor cell growth-inhibitory activities on a panel of four adherent cancer cell lines, and different
Introduction !
Artemisia species (Asteraceae) are of great potential for providing exciting lead compounds for drug discovery and development. With the success of the Artemisia annua metabolite artemisinin in the therapy of malaria, Artemisia species have attracted increasing interest, especially as concern the sesquiterpene constituents of the genus. Besides an antimalarial effect, Artemisinin and its derivatives, which display relatively low toxicity in humans, also exert cytotoxic activity by inducing apoptosis in human cancer cells [1]. Many Artemisia species have been used in traditional medicine for the treatment of different carcinomas, scleromas, and indurations of the uterus, stomach, liver, spleen, and limbs [2], but the majority of Artemisia species have not been investigated for their cytotoxic activity, and the com-
Hajdú Z et al. Antiproliferative Activity of …
Planta Med 2014; 80: 1692–1697
types of secondary metabolites were found to be responsible for the cytotoxic effects of the extract. Especially cirsilineol (13), 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en12,6α-olide (3), and iso-seco-tanapartholide 3-Omethyl ester (5) exerted marked cytotoxic effects against the investigated cell lines, while jaceosidin (12), 6-methoxytricin (15), artecanin (2), and 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxyflavone (14) were moderately active. All the sesquiterpenes and monoterpenes are reported here for the first time from this species, and in the case of artecanin (2), 3α-chloro-4β,10α-dihydroxy-1β, 2β-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide (4), ridentin (6), and ridentin B (7), previously unreported NMR spectroscopic data were determined. Supporting information available online at http://www.thieme-connect.de/products
pounds responsible for the cytotoxic activity have not been identified. Artemisia asiatica Nakai is a plant native to Asia, common in Korea, China, and Japan, which has been used in traditional oriental medicine for the treatment of cancer, inflammation, and other disorders. Its formulated EtOH extract (DA-9601) has marked antioxidant and anti-inflammatory activities and exhibits cytoprotective effects against experimentally-induced gastrointestinal, hepatic, and pancreatic damage [3]. The inhibitory effects of this standardized extract on phorbol ester-induced ornithine decarboxylase activity, papilloma formation, COX-2 expression, inducible nitric oxide synthase expression, and nuclear transcription factor kappa B activation in mouse skin have also been established [4]. In contrast with the very promising pharmacological properties, the chemistry of A. asiatica
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Original Papers
has not been studied thoroughly from the aspect of bioactive constituents, and activity-guided fractionation designed to identify the compounds responsible for the cytotoxic activity has not been performed previously. In the present study, the extract of A. asiatica herb with antiproliferative activity against three human tumor cell lines (HeLa, A431, and MCF7) was analyzed, and bioassay-directed fractionation was carried out in order to obtain more information about the cytotoxic constituents of this species. Sesquiterpene lactones (1–7), monoterpenes (8, 9), and fla" Fig. 1) were isolated from the A. asiatica exvonoids (10–16) (l tract and the tumor cell proliferation-inhibitory activities of the pure compounds were evaluated in vitro.
Chemical structures of compounds 1–16.
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Fig. 1
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Results and Discussion !
In the course of searching for antiproliferative metabolites from the family Asteraceae, n-hexane, CHCl3, H2O-MeOH, and H2O extracts of the flowers, leaves, and stems of A. asiatica were assayed on cervix epithelial adenocarcinoma (HeLa), breast epithelial adenocarcinoma (MCF-7), and skin epidermoid carcinoma (A431) cell lines in vitro. The CHCl3-soluble extract of the flowers and leaves exhibited high cell growth-inhibitory activities at 10 µg/ mL against HeLa (68.60 ± 1.55 and 33.55 ± 0.98 % for the flowers and leaves, respectively), MCF-7 (75.70 ± 1.98 and 57.92 ± 1.83 % for flowers and leaves, respectively), and A431 cells (75.26 ± 1.31 and 72.46 ± 1.41 % for the flowers and leaves, respectively), and the aboveground parts of the plant were therefore subjected to detailed phytochemical-pharmacological analysis [5].
Hajdú Z et al. Antiproliferative Activity of …
Planta Med 2014; 80: 1692–1697
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Original Papers
a
13
C NMR data of compounds 2, 6, and 7 [125 MHz (13C), δ (ppm)].
Position
2a
6b
7c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
77.2 56.5 57.5 70.8 43.0 82.3 44.3 22.5 37.9 72.1 139.4 170.5 118.7 27.8 19.7
74.0 41.5 73.5 147.1 120.8 79.9 48.1 25.9 n. d. 149.9 140.3 169.9 118.4 11.6 110.3
76.8 41.2 70.4 150.1 51.3 81.1 51.0 22.3 37.0 44.1 141.1 172.8 117.4 12.1 107.0
In CDCl3; b in DMSO‑d6; c MeOH‑d4; n. d. = not detected
In the preparative experiment, the aerial parts of A. asiatica were extracted with MeOH at room temperature and, after concentration, the extract was partitioned between n-hexane, CHCl3, and H2O. The CHCl3 extract was fractionated in consecutive chromatographic steps under the guidance of antiproliferative assays. Open-column chromatography was first carried out on polyamide with the elution of H2O–MeOH mixtures by guidance of an antiproliferative test, which resulted in the separation of five fractions with different compositions. Multistep chromatography of the active fractions, including CC, CPC, preparative TLC, and gel-filtration, guided by a bioassay, led to the isolation of compounds 1–9 from fraction I, and compounds 10–16 from fractions II and III. The structures were determined by extensive spectroscopic analysis, including 1D and 2D NMR (1H-1H COSY, HSQC, and HMBC), APCIMS, ESIMS, and HRESIMS experiments. Compound 1 was isolated as a colorless amorphous solid with [α]27 D + 1 (c, 0.2, CHCl3). Its ESIMS spectrum showed a molecular ion peak at m/z 297 [M + H]+ and fragment ions at m/z 279 and 261, derived from the sequential loss of 2×H2O. The 1H‑NMR and JMOD (J-modulated Spin-Echo experiment) spectra indicated a highly oxygenated guaianolide sesquiterpene, which was further studied by 1H-1H COSY, HSQC, and HMBC measurements. 2D NMR analysis allowed for the elucidation of the planar structure as 1,2-epoxy-3,4,10-trihydroxyguaianolide for compound 1. A search of the literature data for this structure revealed that the 1 H- and 13C- NMR data of 1 are in good agreement with those reported earlier for artecanin hydrate, a compound first isolated from Artemisia rutifolia and Artemisia laciniata [6]. This compound was later reported from the Iranian species Artemisia deserti, and it was proposed that the name of the compound should be changed to 10-epiajafinin on a structural basis [7]. However, our detailed stereochemical analysis confirmed the 3-epimeric structure as a result of the following NOESY correlations: Starting from the reference point, H-5α at the ring junction, cross-peaks between H-5/H-15, H-5/H-7, H-15/H-3, H-7/H-8a, and H-7/H-9b proved the α-orientation of these protons. NOE effects observed between H-8b/H-14, H-9a/H-14. H-14/H-6, H-6/H-8b, and H-14/ H-2 indicated their β-orientation. Moreover, weak nuclear Overhauser effects between the 15-methyl and 10-OH groups corroborated their α-position. In conclusion, the 3β,4β,10α-trihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide structure was elucidated for 1, supported primarily by the significant
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Planta Med 2014; 80: 1692–1697
Table 2 NMR data of compound 8 [500 MHz (1H), 125 MHz (13C), δ (ppm), (J = Hz), in MeOH‑d4]. Position
8 1
1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b
H
4.93 d (16.5) 4.96 d (10.2) 6.01 dd (16.5, 10.2) – 4.08 d (10.0) 5.21 d (10.0) 1.72 s 1.67 s 1.05 s 0.99 s 4.25 d (7.9) 3.18 m 3.32 m 3.32 m 3.14 m 3.75 dd (11.9, 5.1) 3.65 dd (11.9, 5.1)
13
C
112.4 146.6 42.9 85.9 125.0 136.0 26.4 18.8 24.5 23.3 105.0 75.7 78.3 71.4 77.7 62.7
NOE cross-peak between H-3 and H-15, and only very weak NOE between 3-OH and H-15. In earlier publications, the α-position of H-3 was postulated in consequence of the coupling constant J2,3 = ~ 3 Hz (0.8 Hz for the opposite structure) [8], but this small difference may not be diagnostic in the case of a five-membered ring. All of the above evidence confirmed the structure of this compound as depicted in structural formula 1. Compound 2 was found to be identical in all of its spectral characteristics with artecanin [9]. Our 2D NMR analysis led to unam" Table 1), which are published biguous 13C NMR assignments (l here for the first time. Chlorinated guaianolides, 3β-chloro4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en-12,6αolide (3), and 3α-chloro-4β,10α-dihydroxy-1β,2β-epoxy-5α,7αHguai-11(13)-en-12,6α-olide (4), reported earlier from some Achillea and Artemisia species, were identified from A. asiatica on the basis of a comparison of spectroscopic characteristics with published data [9, 10]. Similarly, the NMR data on compound 5 were found to be identical with those of the ring B-opened guaianolide sesquiterpene, iso-seco-tanapartholide 3-O-methyl ester [7, 11]. The germacrane lactone ridentin (6) was also identified " Table from A. asiatica on the basis of the spectroscopic data (l 1) [12]. This compound may play a precursor role in the biosynthetic pathway of eudesmanolides and guaianolides of the genus. In addition, the eudesmanolide ridentin B (7) was identified, and the 13C‑NMR data were assigned through the 2D NMR analysis " Table 1). (l Compound 8 was isolated as a colorless oil with [α]27 D − 34 (c, 0.2, MeOH). It was shown by HRESIMS to have the molecular composition C16H28O6, with the quasimolecular ion peak being observed at m/z 339.1784 [M + Na]+ (calcd. 339.1778). In accordance with this, the 1H‑NMR and JMOD spectra of 8 indicated the presence of a glucose unit (δH 4.25 d, 3.18 m, 2 × 3.32 m, 3.14 m, 3.75 dd, and 3.65 dd; δC 105.0, 75.7, 78.3, 71.4, 77.7, and 62.7), and a " Table 2). C10 monoterpene structural moiety in the molecule (l The HSQC spectrum revealed that the C10 moiety is composed of four methyl, one methylene, and three methine groups, as well as two quaternary carbons. The 1H-1H COSY spectrum defined two structural fragments with correlated protons: CH2=CH– (δH
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Table 1
Original Papers
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Table 3 Antiproliferative effects of the isolated active compounds (IC50, µM) on different human tumor cell lines. Calculated IC50 values (µM) ± SDa HeLa
MCF7
A431
Artecanin (2) 3β-Chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αHguai-11(13)-en-12,6α-olide (3) Iso-seco-tanapartholide 3-O-methyl ester (5) Ridentin (6) Hispidulin (11) Jaceosidin (12) Cirsilineol (13) 5,7,4′,5′-Tetrahydroxy-6,3′-dimethoxyflavone (14) 6-Methoxytricin (15) Chrysoplenetin (16) Cisplatin
21.76 ± 0.81 11.46 ± 0.07
10.18 ± 0.62 13.04 ± 0.65
18.30 ± 0.40 12.07 ± 0.23
7.10 ± 0.98 7.65 ± 0.48
12.04 ± 1.73 inactiveb 5.68 ± 0.44* 23.58 ± 3.53 15.24 ± 0.62 2.25 ± 0.10 24.19 ± 1.74 inactive 12.43 ± 1.48
9.39 ± 1.34 28.09 ± 1.78 inactive* 17.84 ± 2.67 11.37 ± 1.13 23.34 ± 3.46 22.47 ± 2.70 28.20 ± 3.62 9.63 ± 1.06
6.77 ± 0.20 inactive inactive* 16.69 ± 0.48 15.45 ± 1.31 17.55 ± 2.05 20.37 ± 1.60 22.00 ± 1.07 2.84 ± 0.86
4.71 ± 0.35 inactive 14.76 ± 2.19 12.13 ± 1.73 7.62 ± 0.54 9.81 ± 0.51 9.46 ± 0.68 23.52 ± 1.06 1.70 ± 0.55
A2780
Mean values from two determinations with five parallel wells; b Inactive indicates an IC50 value higher than 30 µM; Compounds declared inactive against all four cell lines are not
presented; * Published in ref. [23]
4.93 d, 4.96 d, and 6.01 ddd) and –CH(OR)–CH= (δH 4.08 d and 5.21 d). The HMBC spectrum provided information on the connection of the structural parts and quaternary carbons by longrange correlations between C-3 (δC 42.9) and H-2 (δH 6.01 ddd), H-1 (δH 4.93 d, 4.96 d), H-4 (δH 4.08 d), H-9 (δH 1.05 s), and H-10 (δH 0.99 s), and between C-6 (δC 136.0) and H-4 (δH 4.08 d), H-7 (δH 1.72 s), and H-8 (δH 1.67 s). The connection of the glucose part was confirmed by the HMBC cross-peak between C-1′ (δC 105.0) and H-4 (δH 4.08 d). This evidence was used to propose the structure of artemisia alcohol glucoside for compound 8 as a new natural product. Compound 9 was identical in all of its spectral characteristics with dehydrolinalool oxide [13]. Furthermore, flavonoids were isolated in pure form from the active fractions II and III and identified as eupatilin (10), hispidulin (11), jaceosidin (12), cirsilineol (13), 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxy-flavon (14), 6-methoxytricin (15), and chrysoplenetin (16) [14]. Some of the isolated compounds were subjected to an antiproliferative assay on human adherent cell lines (HeLa, A431, MCF7, " Table 3). Two of the four isolated guaianolides (1 and A2780; l and 4) were ineffective (i.e., their calculated IC50 values were higher than 30 µM) against all of the applied cell lines, while the other two (2 and 3) exhibited considerable antiproliferative properties, especially against ovarian cell line A2780. The growth-inhibitory capacity of seco-guaianolide iso-seco-tanapartholide 3-O-methyl ester (5) was also noteworthy. The two further sesquiterpenes, ridentin (6) and ridentin B (7), and the two monoterpenes artemisia alcohol glucoside (8) and dehydrolinalool oxide (9), did not demonstrate any appreciable action. In regards to the isolated flavonoids (10–16), eupatilin (10) proved to be ineffective against this cell line panel, while most of the others exhibited some relevant cytotoxic action against at least " Table 3). one cell line (l Previous phytochemical studies on A. asiatica revealed the presence of terpenes and flavonoids [14]. The chemical constituents of the essential oil have also been investigated [15], and the antimicrobial assays of the essential oil components revealed the antibacterial and antifungal activity of the monoterpene alcohol fraction and the main constituents 1,8-cineole and selin-11-en4α-ol [16]. Earlier studies have demonstrated that A. asiatica contains a high amount of eupatilin (10), a flavone which exhibits cytotoxic activity through the induction of cell cycle arrest and the
differentiation of gastric carcinoma and endometrial cancer cells [17]. Further, eupatilin has been shown to possess an antimetastatic effect against a human gastric cancer cell line [18]. Another bioactive compound isolated from A. asiatica is the sesquiterpene-monoterpene lactone artemisolide [19]. This compound has been demonstrated to exhibit in vitro cytotoxic activity, with GI50 values of 2–8 µM against cancer cell lines [20]. In the present study, highly oxygenated guaianolide sesquiterpenes [3β,4β,10α-trihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)en-12,6α-olide (1), artecanin (2)], chlorinated guaianolides [3βchloro-4α,10α ‑dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en12,6α-olide (3), and 3α-chloro-4β,10α-dihydroxy-1β,2β-epoxy5α,7αH-guai-11(13)-en-12,6α-olide (4)], the seco-guaianolide iso-seco-tanapartholide methyl ester (5), the germacranolide ridentin (6), the eudesmanolide ridentin B (7), and the essential oil component artemisia alcohol in the glucosylated form (8) were isolated from the herb of A. asiatica. All the terpenes were detected in this plant for the first time, and artemisia alcohol glucoside was identified as a new natural product. 13C‑NMR data of " Table 1) and 1H‑NMR data of comcompounds 2, 6, and 7 (l pound 2 in MeOH-d4, compound 4 in acetone-d6 (Supporting In" Table 1) are pubformation), and compound 6 in DMSO-d6 (l lished for the first time in this paper. The isolation procedure under the guidance of the antiproliferative assay led to the conclusion that the bioactive compounds are flavonoids and sesquiterpenes. The pronounced inhibitory effect of the CHCl3 extract of A. asiatica on the proliferation of adherent human tumor cell lines may be attributed mainly to flavonoids [hispidulin (11), jaceosidin (12), cirsilineol (13), 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxyflavone (14), 6-methoxytricin (15), and chrysoplenetin (16)], guaianolides (2, 3), and seco-guaianolide sesquiterpenes (5). While most of the tested natural products exhibited similar activities against the utilized cell lines, two compounds, 11 and 14, inhibited the proliferation of HeLa cells selectively. Though the tested active components may directly contribute to the overall efficacy of the effective extract, a synergistic interaction between the components cannot be excluded.
Hajdú Z et al. Antiproliferative Activity of …
Planta Med 2014; 80: 1692–1697
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a
Compound
Original Papers
Materials and Methods !
General experimental procedures Melting points are uncorrected. Column chromatography (CC) was carried out on polyamide (ICN), preparative thin-layer chromatography (TLC) on silica gel 60 F254 and RP-18 F254 plates (Merck), and centrifugal planar chromatography (CPC) on silica gel 60 GF254 with a Chromatotron instrument (Harrison Research). NMR spectra were recorded on a Bruker Avance DRX 500 spectrometer at 500 MHz for 1H and at 125 MHz for 13C; the signals of the deuterated solvents were taken as the references. Two-dimensional experiments (1H-1H COSY, HSQC, HMBC, and NOESY) were set up, performed, and processed with the standard Bruker protocol. HRESIMS was carried out on an Applied Biosystems 3200 QTrap instrument in ion trap mode. The sample was injected into an acetonitrile flow; the flow rate was 200 mL/min. APCIMS and ESIMS measurements were performed on an API 2000 LC‑MS system.
Plant material A. asiatica Nakai was gathered in September 2008 in the experimental field of the Institute of Ecology and Botany of the Hungarian Academy of Sciences, Vácrátót, Hungary. The plant material was identified by I. Máthé (Institute of Ecology and Botany, Centre for Ecological Research, Hungarian Academy of Sciences, Vácrátót, Hungary). A voucher specimen has been deposited at the herbarium of the Institute of Pharmacognosy, University of Szeged; voucher no. 703.
Extraction, fractionation, and isolation procedures Dried and powdered aerial parts (2223 g) of A. asiatica were extracted with MeOH (45 L) and evaporated to dryness to give 464 g of residual dried extract. This was then dissolved in H2O–MeOH (1 : 1) (400 mL) and partitioned first with n-hexane (3 × 2 L), and then with CHCl3 (3 × 2 L). Combined CHCl3 extracts were evaporated under reduced pressure to give a residue of 100.6 g. The CHCl3 fraction displayed considerable activity in the antiproliferative assay, and was subjected to separation on a polyamide (MP Polyamid EcoChrom™, 55 × 20 cm) column using H2O–MeOH 8 : 2, 6 : 4, 5 : 5, 4 : 6, and 2 : 8 mixtures and MeOH as eluents (2.5 L of each), which afforded fractions I–V. Monitoring of the activities of the fractions indicated that the cell proliferation inhibitory activity was located in three fractions, which were highly effective against A431 cells (inhibition at 30 µg/mL: 61.62 ± 0.66 % for fraction I, 67.66 ± 1.34 % for fraction II, and 98.04 ± 0.37 % for fraction III). Fraction I obtained with MeOH–H2O 2 : 8 was subjected to separation by vacuum liquid chromatography on a silica gel (14 × 8.5 cm, 15 µm, Merck) column (S1) with CHCl3–MeOH mixtures of increasing polarity as eluents collecting 95 × 100 mL fractions. The eluates were combined, depending on their TLC profile. The combined fractions obtained with CHCl3–MeOH 98 : 2 as the eluent were further chromatographed on TLC, using silica gel (Kieselgel 60 GF25415 µm, Merck) as the stationary phase and CHCl3–MeOH 98 : 2 as the eluent. Six bands were removed from the plates, affording pure compounds 2 (13.9 mg), 3 (41.5 mg), 4 (3.0 mg), and 5 (98.6 mg). From the fractions of the S1 column eluted with CHCl3–MeOH 96 : 4 as the eluent, compound 9 was isolated on silica gel (aluminiumoxid 60 G neutral Typ E Merk) CPC with cyclohexane–EtOAc–EtOH solvent systems with increasing polarity; the volume of the eluted fractions was 5 ml. It was then isolated by TLC (Kieselgel 60 GF254, Merck) using solvent system n-hexane–EtOAc–EtOH 7 : 3 : 0.5. From the fractions
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Planta Med 2014; 80: 1692–1697
of the S1 column eluted with CHCl3–MeOH 94 : 6 as the eluent, compound 6 (50.0 mg) was crystallized, further purified by recrystallization, and 7 (14.0 mg) was additionally isolated by preparative TLC on silica gel using toluene–EtOAc–HCOOH (5 : 4 : 1) as the developing system. Two fractions of the S1 column, obtained with CHCl3–MeOH (9 : 1), were further purified. One of them was further separated by CPC [with alumina (aluminiumoxid 60 G neutral Typ E Merk) as the sorbent and CHCl3–MeOH (98 : 2)] as the eluent, and then eluted on a Sephadex LH 20 (Pharmacia) column with MeOH as the eluent, which yielded compound 8 (46.0 mg). The other S1 fraction was separated by preparative TLC on silica gel 60 RP-18, with H2O–MeOH 4 : 6 as the eluent. By this means, pure compound 1 (5.9 mg) was obtained. Isolation and structure elucidation of seven flavonoids [eupatilin (10), hispidulin (11), jaceosidin (12), cirsilineol (13), 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxyflavone (14), 6-methoxytricin (15), and chrysosplenetin (16)] from fractions II and III, involved in the antiproliferative assay, were described previously [14]. 3β,4β,10α-Trihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en1 12,6α-olide (1): a colorless oil; [α]27 D + 1 (c, 0.2, CHCl3); H- and 13 C‑NMR data are identical with those published for artecanin hydrate [6]; ESIMS m/z 314 [M + NH4]+, 297 [M + H]+, 279 [(M + H) – H2O]+, 261 [(M + H) – 2 × H2O]+. 1 Artecanin (2): white needles; [α]25 D + 43 (c, 0.1, MeOH); H‑NMR: 13 " see Supporting Information; C‑NMR: see l Table 1. 3β-Chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)en-12,6α-olide (3): white needles; 1H- and 13C‑NMR: the data were identical with those reported in ref. [10]. 3α-Chloro-4β,10α-dihydroxy-1β,2β-epoxy-5α,7αH-guai-11(13)en-12,6α-olide (4): a white amorphous powder; 1H‑NMR: see Supporting Information; 13C‑NMR: the data were identical with those published in ref. [9]. Iso-seco-tanapartholide methyl ester (5): a yellow oil; the 1H- and 13 C‑NMR: data were identical with those reported in refs. [6, 11]. Ridentin (6): white crystals; 1H‑NMR: see Supporting Infor" Table 1; APCIMS m/z 306 [(M + H) + mation; 13C‑NMR: see l + + MeCN] , 265 [M + H] , 247 [(M + H) – H2O]+, 229 [(M + H) – 2 × H2O]+, 211 [(M + H) – 3 × H2O]+, 188, 183, 173. Ridentin B (7): white needles; m. p. 202–204 °C; APCI‑MS: m/z 282 [M + NH4]+, 265 [M + H]+, 247 [(M + H) – H2O]+, 229 [(M + H) – 2 × H2O]+; 1H‑NMR: the data were identical with those pub" Table 1. lished in ref. [21]; 13C‑NMR (125 MHz, CD3OD): see l 27 Artemisia alcohol glucoside (8): a colorless oil; [α]D − 34 (c, 0.2, " Table 1; HRESIMS m/z MeOH); 1H- and 13C‑NMR: data: see l 339.1784 [M + Na]+ (calcd. for C16H28O6, 339.1778). Dehydrolinalool oxide (9): a colorless oil; [α]25 D − 4 (c 0.15, CHCl3); the 1H- and 13C‑NMR data were found to be identical with those reported for dehydrolinalool oxide [18].
Antiproliferative assay Antiproliferative effects were measured in vitro on HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma), A431 (skin epidermoid carcinoma), and A2780 (ovarian carcinoma) cells, using the MTT assay. All cell lines were purchased from the European Collection of Cell Cultures and maintained in minimal essential medium supplemented with 10% FBS, 1% nonessential amino acids, and an antibiotic-antimycotic mixture (AAM). All experiments were carried out in duplicate on 96-well microplates with at least five parallel wells. Stock solutions of 10 mg/ mL of the tested extracts and 10 mM of compounds were prepared with DMSO. Cisplatin (Ebewe Pharma), an agent adminis-
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tered clinically in certain malignancies, was used as a positive control. The purity of the isolated compounds tested in the bioassay was over 95% with the exception of 5,7,4′,5′-tetrahydroxy6,3′-dimethoxyflavone (14), in which case the purity was 81%. The assays were performed as published previously [22, 23]. Briefly, cells were seeded onto 96-well plates at a density of 5000 cells/well and allowed to stand overnight, after which the medium containing the tested compound was added. After a 72h incubation, viability was determined by the addition of 20 µL of MTT solution (5 mg/mL). The precipitated formazan crystals were solubilized in DMSO, and the absorbance was determined at 545 nm with an ELISA reader. The IC50 values were determined in the concentration range 0.3–30 µg/mL, and concentration-response curves were fitted by means of the computer program GraphPad Prism 4.03.
5
6 7
8
9
10
Supporting information 1
H‑NMR data for compounds 2, 4, and 6 are available as Supporting Information.
11 12
Acknowledgments
13
!
14
This research was supported by the New Hungary Development Plan project TÁMOP‑4.2.2.A-11/1/KONV-2012–0035. Financial support from the Hungarian Scientific Research Fund (OTKA K109846 and OTKA K109293) is gratefully acknowledged. The authors are grateful to Mr. Péter Bérdi for his valuable contribution to the antiproliferative assays.
15 16 17
Conflict of Interest !
18
There are no conflicts of interest for any of the authors. 19
References 1 Morrissey C, Gallis B, Solazzi JW, Kim BJ, Gulati R, Vakar-Lopez F, Goodlett DR, Vessella RL, Sasaki T. Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells. Anticancer Drugs 2010; 21: 423– 432 2 Hartwell JL. Plants used against cancer. A survey (Review). Lloydia 1968; 31: 71–170 3 Baek YH, Lee KN, Jun DW, Yoon BC, Kim JM, Oh TY, Lee OY. Augmenting effect of DA-9601 on ghrelin in an acute gastric injury model. Gut Liver 2011; 5: 52–56 4 Seo HJ, Park KK, Han SS, Chung WY, Son MW, Kim WB, Surh YJ. Inhibitory effects of the standardized extract (DA-9601) of Artemisia asiatica Nakai on phorbol ester-induced ornithine decarboxylase activity, papilloma formation, cyclooxygenase-2 expression, inducible nitric oxide
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synthase expression and nuclear transcription factor kappa B activation in mouse skin. Int J Cancer 2002; 100: 456–462 Réthy B, Csupor-Löffler B, Zupkó I, Hajdú Z, Máthé I, Hohmann J, Rédei T, Falkay G. Antiproliferative activity of Hungarian Asteraceae species against human cancer cell lines. Part I. Phytother Res 2007; 21: 1200– 1208 Huneck S, Zdero C, Bohlmann F. Seco-guaianolides and other constituents from Artemisia species. Phytochemistry 1986; 25: 883–889 Marco JA, Sanz-Cervera JF, Manglano E, Sancenon F, Rustaiyan A, Kardar M. Sesquiterpene lactones from iranian Artemisia species. Phytochemistry 1993; 34: 1561–1564 Trifunović S, Milosavljević S, Vajs V, Macura S, Todorović N. Stereochemistry and conformations of natural 1,2-epoxy-guaianolides based on 1D and 2D NMR data and semiempirical calculations. Magn Reson Chem 2008; 46: 427–431 Trifunović S, Vajs V, Juranić Z, Žižak Z, Tešević V, Macura S, Milosavljević S. Cytotoxic constituents of Achillea clavennae from Montenegro. Phytochemistry 2006; 67: 887–893 Trifunovic S, Aljančić I, Vajs V, Macura S, Milosavljević S. Sesquiterpene lactones and flavonoids of Achillea depressa. Biochem Syst Ecol 2005; 33: 317–322 Tan RX, Jia JZ, Jakupovic J, Bohlmann F, Huneck S. Sesquiterpene lactones from Artemisia rutifolia. Phytochemistry 1991; 30: 3033–3035 Irwin MA, Lee KH, Simson RF, Geisman TA. Sesquiterpene lactones of Artemisia. Ridentin. Phytochemistry 1969; 8: 2009–2012 Asaruddin MR, Honda G, Tsubouchi A, Shimada JN, Aoki T, Kiuchi F. Trypanocydal constituents from Michelia alba. Nat Med 2003; 57: 61–65 Hajdú Z, Martins A, Orbán-Gyapai O, Forgo P, Jedlinszki N, Máthé I, Hohmann J. Xanthine oxidase inhibitory activity and antioxidant properties of extract and flavonoids of Artemisia asiatica. Rec Nat Prod 2014; 8: 299–302 Kalemba D. Constituents of the essential oil of Artemisia asiatica Nakai. Flavour Frag J 1999; 14: 173–176 Kalemba D, Kusewicz D, Świąder K. Antimicrobial properties of the essential oil of Artemisia asiatica Nakai. Phytother Res 2002; 16: 288–291 Oh TY, Shin CY, Sohn YS, Kim DH, Ahn BO, Lee EB, Park CH. Therapeutic effect of DA-9601 on chronic reflux gastritis induced by sodium taurocholate in rats. World J Gastroenterol 2005; 11: 7430–7435 Park BB, Yoon JS, Kim ES, Choi J, Won YW, Choi JH, Lee YY. Inhibitory effects of eupatilin on tumor invasion of human gastric cancer MKN‑1 cells. Tumor Biol 2013; 34: 875–885 Reddy AM, Lee JY, Seo JH, Kim BH, Chung EY, Ryu SY, Kim YS, Lee CK, Min KR, Kim Y. Artemisolide from Artemisia asiatica, nuclear factor-kappaB inhibitor suppressing prostaglandin E2 and nitric oxide production in macrophages. Arch Pharm Res 2006; 29: 591–597 Kim JH, Kim H, Jeon SB, Son K, Kim EH, Kang SK, Sung N, Kwon BM. New sesquiterpene-monoterpene lactone, artemisolide, isolated from Artemisia argyi. Tetrahedron Lett 2002; 43: 6205–6208 Irwin MA, Geismann TA. Ridentin B: an eudesmanolide from Artemisia tripartita ssp. rupicola. Phytochemistry 1973; 12: 871–873 Csupor-Löffler B, Hajdú Z, Zupkó I, Molnár J, Forgo P, Vasas A, Kele Z, Hohmann J. Antiproliferative constituents of the roots of Conyza canadensis. Planta Med 2011; 77: 1183–1188 Forgo P, Zupkó I, Molnár J, Vasas A, Dombi G, Hohmann J. Bioactivityguided isolation of antiproliferative compounds from Centaurea jacea L. Fitoterapia 2012; 83: 921–925
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Original Papers
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Antiproliferative Activity of Artemisia asiatica Extract and Its Constituents on Human Tumor Cell Lines
Authors
Zsuzsanna Hajdú 1, Judit Hohmann 1, Peter Forgo 1, Imre Máthé 1, 2, Judit Molnár 3, István Zupkó 3
Affiliations
1 2 3
Key words " Artemisia asiatica l " Asteraceae l " antiproliferative activity l " sesquiterpene lactones l " monoterpenes l " flavonoids l
received revised accepted
May 19, 2014 Sept. 9, 2014 Sept. 15, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1383146 Published online October 8, 2014 Planta Med 2014; 80: 1692–1697 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Judit Hohmann University of Szeged Department of Pharmacognosy Eotvos u. 6 6720 Szeged Hungary Phone: + 36 62 54 64 53 Fax: + 36 62 54 57 04 [email protected]
Institute of Pharmacognosy, University of Szeged, Szeged, Hungary Institute of Ecology and Botany, Centre for Ecological Research, Hungarian Academy of Sciences, Vácrátót, Hungary Institute of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged, Hungary
Abstract !
The extract of Artemisia asiatica herb with antiproliferative activity against four human tumor cell lines (A2780, A431, HeLa, and MCF7) was analyzed by the MTT assay, and bioassay-directed fractionation was carried out in order to identify the compounds responsible for the cytotoxic activity. Guaianolide (1–4), seco-guianolide (5), germacranolide (6) and eudesmanolide sesquiterpenes (7), monoterpenes (8, 9), including the new compound artemisia alcohol glucoside (8), and flavonoids (10–16) were isolated as a result of a multistep chromatographic procedure (CC, CPC, PLC, and gel filtration). The compounds were identified by means of UV, MS, and NMR spectroscopy, including 1H‑and 13C‑NMR, 1H-1H COSY, NOESY, HSQC, and HMBC experiments. The isolated compounds 1–16 were evaluated for their tumor cell growth-inhibitory activities on a panel of four adherent cancer cell lines, and different
Introduction !
Artemisia species (Asteraceae) are of great potential for providing exciting lead compounds for drug discovery and development. With the success of the Artemisia annua metabolite artemisinin in the therapy of malaria, Artemisia species have attracted increasing interest, especially as concern the sesquiterpene constituents of the genus. Besides an antimalarial effect, Artemisinin and its derivatives, which display relatively low toxicity in humans, also exert cytotoxic activity by inducing apoptosis in human cancer cells [1]. Many Artemisia species have been used in traditional medicine for the treatment of different carcinomas, scleromas, and indurations of the uterus, stomach, liver, spleen, and limbs [2], but the majority of Artemisia species have not been investigated for their cytotoxic activity, and the com-
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types of secondary metabolites were found to be responsible for the cytotoxic effects of the extract. Especially cirsilineol (13), 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en12,6α-olide (3), and iso-seco-tanapartholide 3-Omethyl ester (5) exerted marked cytotoxic effects against the investigated cell lines, while jaceosidin (12), 6-methoxytricin (15), artecanin (2), and 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxyflavone (14) were moderately active. All the sesquiterpenes and monoterpenes are reported here for the first time from this species, and in the case of artecanin (2), 3α-chloro-4β,10α-dihydroxy-1β, 2β-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide (4), ridentin (6), and ridentin B (7), previously unreported NMR spectroscopic data were determined. Supporting information available online at http://www.thieme-connect.de/products
pounds responsible for the cytotoxic activity have not been identified. Artemisia asiatica Nakai is a plant native to Asia, common in Korea, China, and Japan, which has been used in traditional oriental medicine for the treatment of cancer, inflammation, and other disorders. Its formulated EtOH extract (DA-9601) has marked antioxidant and anti-inflammatory activities and exhibits cytoprotective effects against experimentally-induced gastrointestinal, hepatic, and pancreatic damage [3]. The inhibitory effects of this standardized extract on phorbol ester-induced ornithine decarboxylase activity, papilloma formation, COX-2 expression, inducible nitric oxide synthase expression, and nuclear transcription factor kappa B activation in mouse skin have also been established [4]. In contrast with the very promising pharmacological properties, the chemistry of A. asiatica
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Original Papers
has not been studied thoroughly from the aspect of bioactive constituents, and activity-guided fractionation designed to identify the compounds responsible for the cytotoxic activity has not been performed previously. In the present study, the extract of A. asiatica herb with antiproliferative activity against three human tumor cell lines (HeLa, A431, and MCF7) was analyzed, and bioassay-directed fractionation was carried out in order to obtain more information about the cytotoxic constituents of this species. Sesquiterpene lactones (1–7), monoterpenes (8, 9), and fla" Fig. 1) were isolated from the A. asiatica exvonoids (10–16) (l tract and the tumor cell proliferation-inhibitory activities of the pure compounds were evaluated in vitro.
Chemical structures of compounds 1–16.
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Fig. 1
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Results and Discussion !
In the course of searching for antiproliferative metabolites from the family Asteraceae, n-hexane, CHCl3, H2O-MeOH, and H2O extracts of the flowers, leaves, and stems of A. asiatica were assayed on cervix epithelial adenocarcinoma (HeLa), breast epithelial adenocarcinoma (MCF-7), and skin epidermoid carcinoma (A431) cell lines in vitro. The CHCl3-soluble extract of the flowers and leaves exhibited high cell growth-inhibitory activities at 10 µg/ mL against HeLa (68.60 ± 1.55 and 33.55 ± 0.98 % for the flowers and leaves, respectively), MCF-7 (75.70 ± 1.98 and 57.92 ± 1.83 % for flowers and leaves, respectively), and A431 cells (75.26 ± 1.31 and 72.46 ± 1.41 % for the flowers and leaves, respectively), and the aboveground parts of the plant were therefore subjected to detailed phytochemical-pharmacological analysis [5].
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a
13
C NMR data of compounds 2, 6, and 7 [125 MHz (13C), δ (ppm)].
Position
2a
6b
7c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
77.2 56.5 57.5 70.8 43.0 82.3 44.3 22.5 37.9 72.1 139.4 170.5 118.7 27.8 19.7
74.0 41.5 73.5 147.1 120.8 79.9 48.1 25.9 n. d. 149.9 140.3 169.9 118.4 11.6 110.3
76.8 41.2 70.4 150.1 51.3 81.1 51.0 22.3 37.0 44.1 141.1 172.8 117.4 12.1 107.0
In CDCl3; b in DMSO‑d6; c MeOH‑d4; n. d. = not detected
In the preparative experiment, the aerial parts of A. asiatica were extracted with MeOH at room temperature and, after concentration, the extract was partitioned between n-hexane, CHCl3, and H2O. The CHCl3 extract was fractionated in consecutive chromatographic steps under the guidance of antiproliferative assays. Open-column chromatography was first carried out on polyamide with the elution of H2O–MeOH mixtures by guidance of an antiproliferative test, which resulted in the separation of five fractions with different compositions. Multistep chromatography of the active fractions, including CC, CPC, preparative TLC, and gel-filtration, guided by a bioassay, led to the isolation of compounds 1–9 from fraction I, and compounds 10–16 from fractions II and III. The structures were determined by extensive spectroscopic analysis, including 1D and 2D NMR (1H-1H COSY, HSQC, and HMBC), APCIMS, ESIMS, and HRESIMS experiments. Compound 1 was isolated as a colorless amorphous solid with [α]27 D + 1 (c, 0.2, CHCl3). Its ESIMS spectrum showed a molecular ion peak at m/z 297 [M + H]+ and fragment ions at m/z 279 and 261, derived from the sequential loss of 2×H2O. The 1H‑NMR and JMOD (J-modulated Spin-Echo experiment) spectra indicated a highly oxygenated guaianolide sesquiterpene, which was further studied by 1H-1H COSY, HSQC, and HMBC measurements. 2D NMR analysis allowed for the elucidation of the planar structure as 1,2-epoxy-3,4,10-trihydroxyguaianolide for compound 1. A search of the literature data for this structure revealed that the 1 H- and 13C- NMR data of 1 are in good agreement with those reported earlier for artecanin hydrate, a compound first isolated from Artemisia rutifolia and Artemisia laciniata [6]. This compound was later reported from the Iranian species Artemisia deserti, and it was proposed that the name of the compound should be changed to 10-epiajafinin on a structural basis [7]. However, our detailed stereochemical analysis confirmed the 3-epimeric structure as a result of the following NOESY correlations: Starting from the reference point, H-5α at the ring junction, cross-peaks between H-5/H-15, H-5/H-7, H-15/H-3, H-7/H-8a, and H-7/H-9b proved the α-orientation of these protons. NOE effects observed between H-8b/H-14, H-9a/H-14. H-14/H-6, H-6/H-8b, and H-14/ H-2 indicated their β-orientation. Moreover, weak nuclear Overhauser effects between the 15-methyl and 10-OH groups corroborated their α-position. In conclusion, the 3β,4β,10α-trihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide structure was elucidated for 1, supported primarily by the significant
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Table 2 NMR data of compound 8 [500 MHz (1H), 125 MHz (13C), δ (ppm), (J = Hz), in MeOH‑d4]. Position
8 1
1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b
H
4.93 d (16.5) 4.96 d (10.2) 6.01 dd (16.5, 10.2) – 4.08 d (10.0) 5.21 d (10.0) 1.72 s 1.67 s 1.05 s 0.99 s 4.25 d (7.9) 3.18 m 3.32 m 3.32 m 3.14 m 3.75 dd (11.9, 5.1) 3.65 dd (11.9, 5.1)
13
C
112.4 146.6 42.9 85.9 125.0 136.0 26.4 18.8 24.5 23.3 105.0 75.7 78.3 71.4 77.7 62.7
NOE cross-peak between H-3 and H-15, and only very weak NOE between 3-OH and H-15. In earlier publications, the α-position of H-3 was postulated in consequence of the coupling constant J2,3 = ~ 3 Hz (0.8 Hz for the opposite structure) [8], but this small difference may not be diagnostic in the case of a five-membered ring. All of the above evidence confirmed the structure of this compound as depicted in structural formula 1. Compound 2 was found to be identical in all of its spectral characteristics with artecanin [9]. Our 2D NMR analysis led to unam" Table 1), which are published biguous 13C NMR assignments (l here for the first time. Chlorinated guaianolides, 3β-chloro4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en-12,6αolide (3), and 3α-chloro-4β,10α-dihydroxy-1β,2β-epoxy-5α,7αHguai-11(13)-en-12,6α-olide (4), reported earlier from some Achillea and Artemisia species, were identified from A. asiatica on the basis of a comparison of spectroscopic characteristics with published data [9, 10]. Similarly, the NMR data on compound 5 were found to be identical with those of the ring B-opened guaianolide sesquiterpene, iso-seco-tanapartholide 3-O-methyl ester [7, 11]. The germacrane lactone ridentin (6) was also identified " Table from A. asiatica on the basis of the spectroscopic data (l 1) [12]. This compound may play a precursor role in the biosynthetic pathway of eudesmanolides and guaianolides of the genus. In addition, the eudesmanolide ridentin B (7) was identified, and the 13C‑NMR data were assigned through the 2D NMR analysis " Table 1). (l Compound 8 was isolated as a colorless oil with [α]27 D − 34 (c, 0.2, MeOH). It was shown by HRESIMS to have the molecular composition C16H28O6, with the quasimolecular ion peak being observed at m/z 339.1784 [M + Na]+ (calcd. 339.1778). In accordance with this, the 1H‑NMR and JMOD spectra of 8 indicated the presence of a glucose unit (δH 4.25 d, 3.18 m, 2 × 3.32 m, 3.14 m, 3.75 dd, and 3.65 dd; δC 105.0, 75.7, 78.3, 71.4, 77.7, and 62.7), and a " Table 2). C10 monoterpene structural moiety in the molecule (l The HSQC spectrum revealed that the C10 moiety is composed of four methyl, one methylene, and three methine groups, as well as two quaternary carbons. The 1H-1H COSY spectrum defined two structural fragments with correlated protons: CH2=CH– (δH
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Table 1
Original Papers
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Table 3 Antiproliferative effects of the isolated active compounds (IC50, µM) on different human tumor cell lines. Calculated IC50 values (µM) ± SDa HeLa
MCF7
A431
Artecanin (2) 3β-Chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αHguai-11(13)-en-12,6α-olide (3) Iso-seco-tanapartholide 3-O-methyl ester (5) Ridentin (6) Hispidulin (11) Jaceosidin (12) Cirsilineol (13) 5,7,4′,5′-Tetrahydroxy-6,3′-dimethoxyflavone (14) 6-Methoxytricin (15) Chrysoplenetin (16) Cisplatin
21.76 ± 0.81 11.46 ± 0.07
10.18 ± 0.62 13.04 ± 0.65
18.30 ± 0.40 12.07 ± 0.23
7.10 ± 0.98 7.65 ± 0.48
12.04 ± 1.73 inactiveb 5.68 ± 0.44* 23.58 ± 3.53 15.24 ± 0.62 2.25 ± 0.10 24.19 ± 1.74 inactive 12.43 ± 1.48
9.39 ± 1.34 28.09 ± 1.78 inactive* 17.84 ± 2.67 11.37 ± 1.13 23.34 ± 3.46 22.47 ± 2.70 28.20 ± 3.62 9.63 ± 1.06
6.77 ± 0.20 inactive inactive* 16.69 ± 0.48 15.45 ± 1.31 17.55 ± 2.05 20.37 ± 1.60 22.00 ± 1.07 2.84 ± 0.86
4.71 ± 0.35 inactive 14.76 ± 2.19 12.13 ± 1.73 7.62 ± 0.54 9.81 ± 0.51 9.46 ± 0.68 23.52 ± 1.06 1.70 ± 0.55
A2780
Mean values from two determinations with five parallel wells; b Inactive indicates an IC50 value higher than 30 µM; Compounds declared inactive against all four cell lines are not
presented; * Published in ref. [23]
4.93 d, 4.96 d, and 6.01 ddd) and –CH(OR)–CH= (δH 4.08 d and 5.21 d). The HMBC spectrum provided information on the connection of the structural parts and quaternary carbons by longrange correlations between C-3 (δC 42.9) and H-2 (δH 6.01 ddd), H-1 (δH 4.93 d, 4.96 d), H-4 (δH 4.08 d), H-9 (δH 1.05 s), and H-10 (δH 0.99 s), and between C-6 (δC 136.0) and H-4 (δH 4.08 d), H-7 (δH 1.72 s), and H-8 (δH 1.67 s). The connection of the glucose part was confirmed by the HMBC cross-peak between C-1′ (δC 105.0) and H-4 (δH 4.08 d). This evidence was used to propose the structure of artemisia alcohol glucoside for compound 8 as a new natural product. Compound 9 was identical in all of its spectral characteristics with dehydrolinalool oxide [13]. Furthermore, flavonoids were isolated in pure form from the active fractions II and III and identified as eupatilin (10), hispidulin (11), jaceosidin (12), cirsilineol (13), 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxy-flavon (14), 6-methoxytricin (15), and chrysoplenetin (16) [14]. Some of the isolated compounds were subjected to an antiproliferative assay on human adherent cell lines (HeLa, A431, MCF7, " Table 3). Two of the four isolated guaianolides (1 and A2780; l and 4) were ineffective (i.e., their calculated IC50 values were higher than 30 µM) against all of the applied cell lines, while the other two (2 and 3) exhibited considerable antiproliferative properties, especially against ovarian cell line A2780. The growth-inhibitory capacity of seco-guaianolide iso-seco-tanapartholide 3-O-methyl ester (5) was also noteworthy. The two further sesquiterpenes, ridentin (6) and ridentin B (7), and the two monoterpenes artemisia alcohol glucoside (8) and dehydrolinalool oxide (9), did not demonstrate any appreciable action. In regards to the isolated flavonoids (10–16), eupatilin (10) proved to be ineffective against this cell line panel, while most of the others exhibited some relevant cytotoxic action against at least " Table 3). one cell line (l Previous phytochemical studies on A. asiatica revealed the presence of terpenes and flavonoids [14]. The chemical constituents of the essential oil have also been investigated [15], and the antimicrobial assays of the essential oil components revealed the antibacterial and antifungal activity of the monoterpene alcohol fraction and the main constituents 1,8-cineole and selin-11-en4α-ol [16]. Earlier studies have demonstrated that A. asiatica contains a high amount of eupatilin (10), a flavone which exhibits cytotoxic activity through the induction of cell cycle arrest and the
differentiation of gastric carcinoma and endometrial cancer cells [17]. Further, eupatilin has been shown to possess an antimetastatic effect against a human gastric cancer cell line [18]. Another bioactive compound isolated from A. asiatica is the sesquiterpene-monoterpene lactone artemisolide [19]. This compound has been demonstrated to exhibit in vitro cytotoxic activity, with GI50 values of 2–8 µM against cancer cell lines [20]. In the present study, highly oxygenated guaianolide sesquiterpenes [3β,4β,10α-trihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)en-12,6α-olide (1), artecanin (2)], chlorinated guaianolides [3βchloro-4α,10α ‑dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en12,6α-olide (3), and 3α-chloro-4β,10α-dihydroxy-1β,2β-epoxy5α,7αH-guai-11(13)-en-12,6α-olide (4)], the seco-guaianolide iso-seco-tanapartholide methyl ester (5), the germacranolide ridentin (6), the eudesmanolide ridentin B (7), and the essential oil component artemisia alcohol in the glucosylated form (8) were isolated from the herb of A. asiatica. All the terpenes were detected in this plant for the first time, and artemisia alcohol glucoside was identified as a new natural product. 13C‑NMR data of " Table 1) and 1H‑NMR data of comcompounds 2, 6, and 7 (l pound 2 in MeOH-d4, compound 4 in acetone-d6 (Supporting In" Table 1) are pubformation), and compound 6 in DMSO-d6 (l lished for the first time in this paper. The isolation procedure under the guidance of the antiproliferative assay led to the conclusion that the bioactive compounds are flavonoids and sesquiterpenes. The pronounced inhibitory effect of the CHCl3 extract of A. asiatica on the proliferation of adherent human tumor cell lines may be attributed mainly to flavonoids [hispidulin (11), jaceosidin (12), cirsilineol (13), 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxyflavone (14), 6-methoxytricin (15), and chrysoplenetin (16)], guaianolides (2, 3), and seco-guaianolide sesquiterpenes (5). While most of the tested natural products exhibited similar activities against the utilized cell lines, two compounds, 11 and 14, inhibited the proliferation of HeLa cells selectively. Though the tested active components may directly contribute to the overall efficacy of the effective extract, a synergistic interaction between the components cannot be excluded.
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Planta Med 2014; 80: 1692–1697
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a
Compound
Original Papers
Materials and Methods !
General experimental procedures Melting points are uncorrected. Column chromatography (CC) was carried out on polyamide (ICN), preparative thin-layer chromatography (TLC) on silica gel 60 F254 and RP-18 F254 plates (Merck), and centrifugal planar chromatography (CPC) on silica gel 60 GF254 with a Chromatotron instrument (Harrison Research). NMR spectra were recorded on a Bruker Avance DRX 500 spectrometer at 500 MHz for 1H and at 125 MHz for 13C; the signals of the deuterated solvents were taken as the references. Two-dimensional experiments (1H-1H COSY, HSQC, HMBC, and NOESY) were set up, performed, and processed with the standard Bruker protocol. HRESIMS was carried out on an Applied Biosystems 3200 QTrap instrument in ion trap mode. The sample was injected into an acetonitrile flow; the flow rate was 200 mL/min. APCIMS and ESIMS measurements were performed on an API 2000 LC‑MS system.
Plant material A. asiatica Nakai was gathered in September 2008 in the experimental field of the Institute of Ecology and Botany of the Hungarian Academy of Sciences, Vácrátót, Hungary. The plant material was identified by I. Máthé (Institute of Ecology and Botany, Centre for Ecological Research, Hungarian Academy of Sciences, Vácrátót, Hungary). A voucher specimen has been deposited at the herbarium of the Institute of Pharmacognosy, University of Szeged; voucher no. 703.
Extraction, fractionation, and isolation procedures Dried and powdered aerial parts (2223 g) of A. asiatica were extracted with MeOH (45 L) and evaporated to dryness to give 464 g of residual dried extract. This was then dissolved in H2O–MeOH (1 : 1) (400 mL) and partitioned first with n-hexane (3 × 2 L), and then with CHCl3 (3 × 2 L). Combined CHCl3 extracts were evaporated under reduced pressure to give a residue of 100.6 g. The CHCl3 fraction displayed considerable activity in the antiproliferative assay, and was subjected to separation on a polyamide (MP Polyamid EcoChrom™, 55 × 20 cm) column using H2O–MeOH 8 : 2, 6 : 4, 5 : 5, 4 : 6, and 2 : 8 mixtures and MeOH as eluents (2.5 L of each), which afforded fractions I–V. Monitoring of the activities of the fractions indicated that the cell proliferation inhibitory activity was located in three fractions, which were highly effective against A431 cells (inhibition at 30 µg/mL: 61.62 ± 0.66 % for fraction I, 67.66 ± 1.34 % for fraction II, and 98.04 ± 0.37 % for fraction III). Fraction I obtained with MeOH–H2O 2 : 8 was subjected to separation by vacuum liquid chromatography on a silica gel (14 × 8.5 cm, 15 µm, Merck) column (S1) with CHCl3–MeOH mixtures of increasing polarity as eluents collecting 95 × 100 mL fractions. The eluates were combined, depending on their TLC profile. The combined fractions obtained with CHCl3–MeOH 98 : 2 as the eluent were further chromatographed on TLC, using silica gel (Kieselgel 60 GF25415 µm, Merck) as the stationary phase and CHCl3–MeOH 98 : 2 as the eluent. Six bands were removed from the plates, affording pure compounds 2 (13.9 mg), 3 (41.5 mg), 4 (3.0 mg), and 5 (98.6 mg). From the fractions of the S1 column eluted with CHCl3–MeOH 96 : 4 as the eluent, compound 9 was isolated on silica gel (aluminiumoxid 60 G neutral Typ E Merk) CPC with cyclohexane–EtOAc–EtOH solvent systems with increasing polarity; the volume of the eluted fractions was 5 ml. It was then isolated by TLC (Kieselgel 60 GF254, Merck) using solvent system n-hexane–EtOAc–EtOH 7 : 3 : 0.5. From the fractions
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of the S1 column eluted with CHCl3–MeOH 94 : 6 as the eluent, compound 6 (50.0 mg) was crystallized, further purified by recrystallization, and 7 (14.0 mg) was additionally isolated by preparative TLC on silica gel using toluene–EtOAc–HCOOH (5 : 4 : 1) as the developing system. Two fractions of the S1 column, obtained with CHCl3–MeOH (9 : 1), were further purified. One of them was further separated by CPC [with alumina (aluminiumoxid 60 G neutral Typ E Merk) as the sorbent and CHCl3–MeOH (98 : 2)] as the eluent, and then eluted on a Sephadex LH 20 (Pharmacia) column with MeOH as the eluent, which yielded compound 8 (46.0 mg). The other S1 fraction was separated by preparative TLC on silica gel 60 RP-18, with H2O–MeOH 4 : 6 as the eluent. By this means, pure compound 1 (5.9 mg) was obtained. Isolation and structure elucidation of seven flavonoids [eupatilin (10), hispidulin (11), jaceosidin (12), cirsilineol (13), 5,7,4′,5′-tetrahydroxy-6,3′-dimethoxyflavone (14), 6-methoxytricin (15), and chrysosplenetin (16)] from fractions II and III, involved in the antiproliferative assay, were described previously [14]. 3β,4β,10α-Trihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en1 12,6α-olide (1): a colorless oil; [α]27 D + 1 (c, 0.2, CHCl3); H- and 13 C‑NMR data are identical with those published for artecanin hydrate [6]; ESIMS m/z 314 [M + NH4]+, 297 [M + H]+, 279 [(M + H) – H2O]+, 261 [(M + H) – 2 × H2O]+. 1 Artecanin (2): white needles; [α]25 D + 43 (c, 0.1, MeOH); H‑NMR: 13 " see Supporting Information; C‑NMR: see l Table 1. 3β-Chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)en-12,6α-olide (3): white needles; 1H- and 13C‑NMR: the data were identical with those reported in ref. [10]. 3α-Chloro-4β,10α-dihydroxy-1β,2β-epoxy-5α,7αH-guai-11(13)en-12,6α-olide (4): a white amorphous powder; 1H‑NMR: see Supporting Information; 13C‑NMR: the data were identical with those published in ref. [9]. Iso-seco-tanapartholide methyl ester (5): a yellow oil; the 1H- and 13 C‑NMR: data were identical with those reported in refs. [6, 11]. Ridentin (6): white crystals; 1H‑NMR: see Supporting Infor" Table 1; APCIMS m/z 306 [(M + H) + mation; 13C‑NMR: see l + + MeCN] , 265 [M + H] , 247 [(M + H) – H2O]+, 229 [(M + H) – 2 × H2O]+, 211 [(M + H) – 3 × H2O]+, 188, 183, 173. Ridentin B (7): white needles; m. p. 202–204 °C; APCI‑MS: m/z 282 [M + NH4]+, 265 [M + H]+, 247 [(M + H) – H2O]+, 229 [(M + H) – 2 × H2O]+; 1H‑NMR: the data were identical with those pub" Table 1. lished in ref. [21]; 13C‑NMR (125 MHz, CD3OD): see l 27 Artemisia alcohol glucoside (8): a colorless oil; [α]D − 34 (c, 0.2, " Table 1; HRESIMS m/z MeOH); 1H- and 13C‑NMR: data: see l 339.1784 [M + Na]+ (calcd. for C16H28O6, 339.1778). Dehydrolinalool oxide (9): a colorless oil; [α]25 D − 4 (c 0.15, CHCl3); the 1H- and 13C‑NMR data were found to be identical with those reported for dehydrolinalool oxide [18].
Antiproliferative assay Antiproliferative effects were measured in vitro on HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma), A431 (skin epidermoid carcinoma), and A2780 (ovarian carcinoma) cells, using the MTT assay. All cell lines were purchased from the European Collection of Cell Cultures and maintained in minimal essential medium supplemented with 10% FBS, 1% nonessential amino acids, and an antibiotic-antimycotic mixture (AAM). All experiments were carried out in duplicate on 96-well microplates with at least five parallel wells. Stock solutions of 10 mg/ mL of the tested extracts and 10 mM of compounds were prepared with DMSO. Cisplatin (Ebewe Pharma), an agent adminis-
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tered clinically in certain malignancies, was used as a positive control. The purity of the isolated compounds tested in the bioassay was over 95% with the exception of 5,7,4′,5′-tetrahydroxy6,3′-dimethoxyflavone (14), in which case the purity was 81%. The assays were performed as published previously [22, 23]. Briefly, cells were seeded onto 96-well plates at a density of 5000 cells/well and allowed to stand overnight, after which the medium containing the tested compound was added. After a 72h incubation, viability was determined by the addition of 20 µL of MTT solution (5 mg/mL). The precipitated formazan crystals were solubilized in DMSO, and the absorbance was determined at 545 nm with an ELISA reader. The IC50 values were determined in the concentration range 0.3–30 µg/mL, and concentration-response curves were fitted by means of the computer program GraphPad Prism 4.03.
5
6 7
8
9
10
Supporting information 1
H‑NMR data for compounds 2, 4, and 6 are available as Supporting Information.
11 12
Acknowledgments
13
!
14
This research was supported by the New Hungary Development Plan project TÁMOP‑4.2.2.A-11/1/KONV-2012–0035. Financial support from the Hungarian Scientific Research Fund (OTKA K109846 and OTKA K109293) is gratefully acknowledged. The authors are grateful to Mr. Péter Bérdi for his valuable contribution to the antiproliferative assays.
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Conflict of Interest !
18
There are no conflicts of interest for any of the authors. 19
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