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Sesquiterpenoids from roots of Lactuca sativa var. angustana cv. “Grüner Stern”
Phytochemistry Letters 20 (2017) 425–428
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Sesquiterpenoids from roots of Lactuca sativa var. angustana cv. “Grüner Stern” Klaudia Michalskaa,* , Oskar Michalskib , Anna Stojakowskaa a b
Institute of Pharmacology, Polish Academy of Sciences, Department of Phytochemistry, Sme˛tna Street 12, 31-343 Kraków, Poland Institute of Chemistry, University of Agriculture in Krakow, Balicka Street 122, 30-149 Kraków, Poland
A R T I C L E I N F O
Article history: Received 5 September 2016 Received in revised form 24 October 2016 Accepted 31 October 2016 Available online 4 November 2016 Keywords: Lactuca sativa var. angustana cv. “Grüner Stern” Asteraceae Sesquiterpene lactones Phenolic compounds
A B S T R A C T
From the roots of Lactuca sativa var. angustana cv. “Grüner Stern” 21 known sesquiterpene lactones, both aglycones and glycosides, together with four phenolic compounds were isolated from ethanol extracts using extensive chromatographic methods (CC, TLC, HPLC/DAD). The compounds were identified by spectroscopic methods (1D- and 2D-NMR) and ESI mass spectrometry. This is the first report on the occurrence of 11b-hydroxyleucodin-11-O-b-glucopyranoside and lactulide B in Lactuca species. ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Stem lettuce, also called asparagus lettuce (Lactuca sativa L. var. angustana L.H. Bailey) is popular in China as both vegetable and medicinal plant (Chinese lettuce, celtuce or “wosun”). Also in Kraków and surrounding area, local cultivars of the plant were once popular as a vegetable (gła˛biki krakowskie). Secondary metabolites synthesized by the plant have not been examined in detail. The only study on sesquiterpenoids from the stalks of a Chinese cultivar of the vegetable (purchased on local market) was published by Han et al. (2010). Sesquiterpene lactones produced by Lactuca ssp. display a vast array of biological activities (Wesołowska et al., 2006; Chadwick et al., 2013) and are deposited in aerial parts as well as in roots of lettuce plants (Zidorn, 2009). The roots, which are not edible, may provide a rich source of the active compounds. Ishihara et al. (1987), from a water extract of L. sativa fresh roots, isolated three sesquiterpene lactone glycosides: lactuside A, lactuside C and macrocliniside A. Our previous comparative HPLC study of eleven Lactuca species, based on distributional data of eight sesquiterpene lactones, revealed that roots of Lactuca sativa L. (cv. British Hilde) contained 8-deoxylactucin, jacquinelin, crepidiaside B, lactucin, 11b,13-dihydrolactucin, lactucopicrin and lactuside A. Moreover, we proved that roots of both cultivated and wild lettuce species are
* Corresponding author. E-mail address: [email protected] (K. Michalska).
richer in sesquiterpenes than aerial parts of the plants (Michalska et al., 2009). Thus, we decided to examine roots of asparagus lettuce cv. “Grüner Stern” in order to assess their usability as a source of sesquiterpene lactones. 2. Results and discussion The present study deals with isolation and identification of 21 known sesquiterpene lactones (Fig. 1, 1–21), together with four phenolic compounds, from roots of L. sativa var. angustana cv. “Grüner Stern”. The known compounds: 11b,13-dihydrolactucin (1), 11b,13dihydrolactucopicrin (2), crepidiaside B (3), cichorioside B (4), lactucin (5), lactucopicrin (6), leucodin (7), 8-deacetylmatricarin8-O-sulfate (8), 11b-hydroxyleucodin-11-O-b-glucopyranoside (9), scorzoside (10), 11b,13-dihydrovernoflexuoside (11), ixerin F (12), macrocliniside A (13), lactuside C (14), 10b,14-dihydroxy-10 (14),11b(13)-tetrahydro-8,9-didehydro-3-deoxyzaluzanin C-10-Ob-glucopyranoside (15), 3b,14-dihydroxy-11b,13-dihydrocostunolide (16), sonchuside A (17), ixerin H (18), picriside B (19), lactulide B (20) lactuside A (21), coniferyl aldehyde, sinapyl aldehyde, eugenyl-4-O-b-glucopyranoside and 3,4,5-trimethoxybenzaldehyde were identified by direct comparison of their spectral data either with those of the reference compounds formerly isolated in our laboratory (Michalska et al., 2014, 2015; Michalska and Kisiel, 2003, 2010, 2013) or with those from the literature (Mahmoud et al., 1986).
http://dx.doi.org/10.1016/j.phytol.2016.10.023 1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
426
K. Michalska et al. / Phytochemistry Letters 20 (2017) 425–428 Table 1 1 H NMR (600.20 MHz) and
13
C NMR (150.94 MHz) data of compound 20 in CDCl3.
Position
dH (ppm), J (Hz)
dC (ppm) HMBC (H ! C)
1 2a 2b 3 4 5 6 7 8a 8b 9a 9b 10 11 12 13 14a 14b 15
5.55 dd (8.1, 8.1) 2.17 ma 2.29 ddd (12.5, 11.3, 8.1) 4.05 dd (11.3, 2.5) – 5.03 br d (9.9) 4.69 dd (9.9, 9.9) 1.58 mb 2.17 ma 1.50 dddd (12.5, 11.5, 5.0, 1.7) 2.17 ma 2.05 ddd (15.0, 15.0, 5.0) – 2.22 dq (12.7, 6.9) – 1.23 d (6.9) 4.03 d (12.7) 4.08 d (12.6) 1.88 d (1.3)
123.28 33.84
a b
76.27 138.09 123.70 80.01 49.14 26.29 23.70 142.07 41.65 178.58 12.92 66.09 10.92
C-2, C-9, C-14 C-1, C-3, C-4 C-1, C-3, C-4 C-1, C-2, C-15 – C-3, C-7, C-15 C-4, C-8 C-5, C-8, C-11 C-6, C-7, C-9, C-10, C-11 C-6, C-7, C-9, C-10, C-11 C-1, C-7, C-8, C-10 C-1, C-7, C-8, C-10, C-14 – C-7, C-8, C-13 – C-7, C-11, C-12 C-1, C-9, C-10 C-1, C-9, C-10 C-3, C-4, C-5
Signals overlapped. Obscured by the signal of H2O.
and 2D NMR measurements, including COSY, NOESY, HMQC and HMBC spectra (Table 1). Analysis of the 1H NMR and 13C NMR spectra indicated that the compound 20 is a melampolide with two double carbon-carbon bonds in the molecule. The position and alpha configuration of the proton at the carbon bearing the hydroxyl group were established on the basis of its signal observed in the 1H NMR spectrum at dH 4.05 (dd, J = 11.3, 2.5 Hz), supported by the COSY and NOESY spectra. Thus a hydroxyl group at C-3 had beta configuration. In the 1H NMR spectrum of 20 two-proton AB systems at dH 4.08 (d, J = 12.6 Hz) and dH 4.03 (d, J = 12.7 Hz) at C-14 could be observed. The presence of an 11b,13-dihydroderivative of a methylene lactone was deduced from the occurrence of a methyl doublet at dH 1.23 (d, J = 6.9 Hz) and a double quartet at dH 2.22 (dq, J = 12.7, 6.9 Hz). The coupling constant of 12.7 Hz indicated an 11bproton. The relative stereochemistry of 20 was determined on the basis of coupling constants of the proton signals, the chemical shifts of the carbon signals as well as the results of NOESY experiments, by making an assumption that H-7 is alpha. The configurations of H-5a, H-6b, H-7a and H-11b were confirmed based on the values of J5a,6b = 9.9 Hz, J6b,7a = 9.9 Hz and J7a,11b = 12.7 Hz, which were indicative of anti-diaxial relationships of the respective protons. More relevant results were NOESY
Fig. 1. Chemical structures of compounds 1–21 (Glc = b-glucopyranosyl, A = 4hydroxyphenylacetyl).
The guaianolide 11b-hydroxyleucodin-11-O-b-glucopyranoside (9) was first obtained from Taraxacum obovatum (Michalska and Kisiel, 2003) and this is the first reported occurrence of 9 in the genus Lactuca. This is also the first report describing the presence of lactulide B (20) in Lactuca spp. The compound was originally isolated from Notoseris rhombiformis and was erroneously identified as 3b,14-dihydroxy-11b,13-dihydrocostunolide, which was not in accordance with the given NMR data taken in CD3OD (Liao et al., 2002). Moreover, the spectral data given by the authors were not complete. We deduced the structure of 20 from its 1H NMR spectrum in CDCl3. The structural assignment was verified by mass Fig. 2. Key NOESY correlations for 20.
K. Michalska et al. / Phytochemistry Letters 20 (2017) 425–428
cross peaks from H-7a to H-3a and H-5a, and from H-6b to H-8b, H-9b, H-11b and H-15. E configuration of the double bond C1-C10 was proved by correlation of protons at C-2 and C-9 in the NOESY spectrum (Fig. 2). ESI mass spectrum of the compound displayed an adduct ion peaks at m/z: 289.0 [M+Na]+ and 555.3 [2M+Na]+ suggesting a molecular formula C15H22O4. Glucoside of 20 (lactuside B) was formerly isolated from roots of Lactuca laciniata Makino (Nishimura et al., 1986). By analogy with lactuside A and its aglycone–lactulide A we named the isolated melampolide (20) lactulide B. The isolated guaianolides: lactucin (5), lactucopicrin (6) and their dihydroderivatives as well as the germacranolide lactuside A (21), are characteristic constituents of cultivated lettuce plants (Tamaki et al., 1995; Sessa et al., 2000; Michalska et al., 2009; AbuReidah et al., 2013). 3b,14-Dihydroxy-11b,13-dihydrocostunolide (16) was found in the aerial parts of L. sativa by Mahmoud et al. (1986). Cichorioside B (4) and 8-deacetylmatricarin-8-O-sulfate (8) have been reported recently (Han et al., 2010; Abu-Reidah et al., 2013; Mai and Glomb, 2016) as constituents of some commercial varieties of lettuce and chicory. According to Tamaki et al. (1995), commercial varieties of lettuce are often devoid of 8-deoxylactucin. This is in agreement with the present study, though we previously found 8-deoxylactucin in roots and aerial parts of L. sativa cv. British Hilde (Michalska et al., 2009). The overall levels of bioactive lactucin-like guaianolides, possessing an exomethylene function, which were found in the commercially grown cultivars of lettuce were usually significantly lower comparing to those in closely related wild species of the genus Lactuca (Tamaki et al., 1995; Michalska et al., 2009; Seo et al., 2009). In the present study isolation yields of lactucin (5) and lactucopicrin (6) were 12.3 mg g1 and 29.8 mg g1, respectively, calculated on the dry weight basis. Macrocliniside A was the only exomethylene-bearing sesquiterpene lactone isolated with relatively high yield (179 mg g1). Han et al. (2010) isolated and identified eight guaianolides and three eudesmanolides from the stalks of commercially available chinese cultivar of L. sativa var. anagustata. Only four of these compounds: 11b,13-dihydrolactucin (1), 11b,13-dihydrolactucopicrin (2), cichorioside B (4) and macrocliniside A (13, isolation yield achieved by Han et al. 2.28 mg g1) were isolated from roots of the plant examined in our laboratory. We did not found any eudesmanolides which were previously described from the Chinese material. However, we isolated and identified six germacranolides (16–21) which were not reported by Han et al. (2010). Compounds 9 and 20 were isolated for the first time from the plant of the genus Lactuca. Lactuside A (21) and 8deacetylmatricarin-8-O-sulfate (8) were among the major sesquiterpene lactones found in the examined roots (isolation yields 160 mg g1 and 210 mg g1, respectively, calculated on a dry weight basis). Our present study reveals diversity of sesquiterpenes produced by closely related cultivars of commercially grown lettuce and usefulness of lettuce by-products as a source of biologically active compounds.
427
with H2O-MeOH mixtures at a flow rate of 3.0 ml min1. Column chromatography was carried out using Merck silica gel 60 (0.063– 0.2 mm). TLC was performed on Merck silica gel 60 (0.25 mm) plates. 3.2. Plant material Roots of L. sativa var. angustana cv. “Grüner Stern” were collected in July 2014 from flowering plants grown in the Garden of Medicinal Plants, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland, where a voucher specimen (03/2014) was deposited. Seeds of the plant were derived from the Botanical Garden of the University of Bonn, Germany. 3.3. Extraction and isolation The dried plant material (228 g) was powdered and exhaustively extracted with ethanol at room temperature with shaking. After removal of the solvent under reduced pressure, the residue (10 g) was passed through a silica gel (Merck, Art. 7754) column using mobile phase gradients of EtOAc in hexane (up to 100% EtOAc), followed by EtOAc-MeOH (up to 5% MeOH). Elution of the column with hexane-EtOAc (7: 3, v/v) and subsequent prep. TLC (hexane-EtOAc, 7: 3, 3: 2, 1: 1) afforded coniferyl aldehyde (3.5 mg), 3,4,5-trimethoxybenzaldehyde (3.7 mg), 7 (12.5 mg) and sinapyl aldehyde (4.1 mg). Fractions from hexane-EtOAc (1: 1) elution furnished 2 (3.4 mg) and 6 (6.8 mg). Further fractions eluted with hexane-EtOAc (1: 1) after separation by prep. TLC (CHCl3-MeOH, 9: 1) and semiprep. HPLC (H2O-MeOH, 7: 3, 3: 2, 1: 1) gave 1 (3.6 mg), 5 (2.8 mg) and 20 (1.9 mg). Fractions eluted with EtOAc, subsequently submitted to prep. TLC (CHCl3-MeOH, 9: 1 or 17: 3) and semiprep. HPLC (H2OMeOH, 3: 1 or 1: 1) yielded 20 (1.6 mg), 16 (1.7 mg), 9 (0.9 mg), 17 and eugenyl-4-O-b-glucopyranoside in a mixture (ca. 4: 1, 1.5 mg), 10 (1.1 mg), 11 and 19 in a mixture (ca. 1: 1.5, 1.6 mg), and 18 (1.2 mg). Less polar fractions eluted with EtOAc-MeOH (49: 1) which were further separated by prep. TLC (CHCl3-MeOH, 17: 3) and semiprep. HPLC (H2O-MeOH, 7: 3) gave 21 (36.8 mg), 14 (21.9 mg), 3 and 15 in a mixture (ca. 1: 5, 5.3 mg) and 15 (6.0 mg). More polar fractions eluted with EtOAc-MeOH (49: 1) and next subjected to prep. TLC (CHCl3-MeOH, 17: 3) gave 12 and 13 in a mixture (ca. 3: 4, 69.1 mg), 8 and 4 in a mixture (ca. 4: 1, 34.8 mg) and 4, 12 and 13 in a mixture (ca. 1: 1: 1, 4.1 mg). 3.4. Lactulide B (20) Colourless solid; ½a26 D 12.6 (c = 0.53, CHCl3); ESIMS (pos. + mode) m/z: 289.0 [M+Na] and 555.3 [2M+Na]+; 1H NMR and 13C NMR: Table 1.
Acknowledgement The financial support of the Ministry of Science and Higher Education, Poland, (statutory activity founding) is gratefully acknowledged.
3. Experimental References 3.1. General experimental procedures All NMR spectra were recorded on a Bruker AVANCE III 400 and a Bruker AVANCE III 600. ESI mass spectra was obtained in the positive ion mode using AmaZon ETD (Bruker-Daltonics). Optical rotation was determined on a PolAAr 31 polarimeter. Semiprep. RP HPLC was performed on a Waters instrument coupled to a dual wavelength UV/VIS detector operating at 210 and 260 nm, using Delta-Pak C-18 column (particle size 15 mm, 25 100 mm) eluted
Abu-Reidah, I.M., Contreras, M.M., Arráez-Román, D., Segura-Carretero, A., Fernández-Gutiérrez, A., 2013. Reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry as a powerful tool for metabolic profiling of vegetables: Lactuca sativa as an example of its application. J. Chromatogr. A 1313, 212–227. Chadwick, M., Trewin, H., Gawthrop, F., Wagstaff, C., 2013. Sesquiterpenoids lactones: benefits to plants and people. Int. J. Mol. Sci. 14, 12780–12805. Han, Y.F., Cao, G.X., Gao, X.J., Xia, M., 2010. Isolation and characterization of the sesquiterpene lactones from Lactuca sativa L. var. anagustata. Food Chem. 120, 1083–1088.
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Ishihara, N., Miyase, T., Ueno, A., 1987. Sesquiterpene glycosides from Lactuca sativa L. Chem. Pharm. Bull. 35, 3905–3908. Liao, Z.-X., Wang, M.-K., Peng, S.-L., Chen, Y.-Z., Ding, L.-S., 2002. Chemical constituents of Notoseris rhombiformis. Acta Pharm. Sin. 37, 37–40. Mahmoud, Z.F., Kassem, F.F., Abdel-Salam, N.A., Zdero, C., 1986. Sesquiterpene lactones from Lactuca sativa. Phytochemistry 25, 747–748. Mai, F., Glomb, M.A., 2016. Structural and sensory characterization of novel sesquiterpene lactones from iceberg lettuce. J. Agric. Food Chem. 64, 295–301. Michalska, K., Kisiel, W., 2003. Sesquiterpene lactones from Taraxacum obovatum. Planta Med. 69, 181–183. Michalska, K., Kisiel, W., 2010. Sesquiterpene lactones from roots of Lactuca aculeata. Biochem. Syst. Ecol. 38, 830–832. Michalska, K., Kisiel, W., 2013. Structural diversity of sesquiterpene lactones in roots of Lactuca viminea. Biochem. Syst. Ecol. 51, 16–18. Michalska, K., Stojakowska, A., Malarz, J., Doležalová, I., Lebeda, A., Kisiel, W., 2009. Systematic implications of sesquiterpene lactones in Lactuca species. Biochem. Syst. Ecol. 37, 174–179. Michalska, K., Beharav, A., Kisiel, W., 2014. Sesquiterpene lactones from roots of Lactuca georgica. Phytochem. Lett. 10, 10–12.
Michalska, K., Kisiel, W., Stojakowska, A., 2015. Chemical constituents of Lactuca dregeana. Biochem. Syst. Ecol. 59, 302–304. Nishimura, K., Miyase, T., Ueno, A., Noro, T., Kuroyanagi, M., Fukushima, S., 1986. Sesquiterpene lactones from Lactuca laciniata. Phytochemistry 25, 2375–2379. Seo, M.W., Yang, D.S., Kays, S.J., Lee, G.P., Park, K.W., 2009. Sesquiterpene lactones and bitterness in Korean leaf lettuce cultivars. HortScience 44, 246–249. Sessa, R.A., Bennett, M.H., Lewis, M.J., Mansfield, J.W., Beale, M.H., 2000. Metabolite profiling of sesquiterpene lactones from Lactuca species. J. Biol. Chem. 275, 26877–26884. Tamaki, H., Robinson, R.W., Anderson, J.L., Stoewsand, G.S., 1995. Sesquiterpene lactones in virus-resistant lettuce. J. Agric. Food Chem. 43, 6–8. Wesołowska, A., Nikiforuk, A., Michalska, K., Kisiel, W., Chojnacka-Wojcik, E., 2006. Analgesic and sedative activities of lactucin and some lactucin-like guaianolides in mice. J. Ethnopharmacol. 107, 254–258. Zidorn, Ch., 2009. Sesquiterpene lactones and their precursors as chemosystematic markers in the tribe Cichorieae of the Asteraceae. Phytochemistry 69, 2270– 2296.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Sesquiterpenoids from roots of Lactuca sativa var. angustana cv. “Grüner Stern” Klaudia Michalskaa,* , Oskar Michalskib , Anna Stojakowskaa a b
Institute of Pharmacology, Polish Academy of Sciences, Department of Phytochemistry, Sme˛tna Street 12, 31-343 Kraków, Poland Institute of Chemistry, University of Agriculture in Krakow, Balicka Street 122, 30-149 Kraków, Poland
A R T I C L E I N F O
Article history: Received 5 September 2016 Received in revised form 24 October 2016 Accepted 31 October 2016 Available online 4 November 2016 Keywords: Lactuca sativa var. angustana cv. “Grüner Stern” Asteraceae Sesquiterpene lactones Phenolic compounds
A B S T R A C T
From the roots of Lactuca sativa var. angustana cv. “Grüner Stern” 21 known sesquiterpene lactones, both aglycones and glycosides, together with four phenolic compounds were isolated from ethanol extracts using extensive chromatographic methods (CC, TLC, HPLC/DAD). The compounds were identified by spectroscopic methods (1D- and 2D-NMR) and ESI mass spectrometry. This is the first report on the occurrence of 11b-hydroxyleucodin-11-O-b-glucopyranoside and lactulide B in Lactuca species. ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Stem lettuce, also called asparagus lettuce (Lactuca sativa L. var. angustana L.H. Bailey) is popular in China as both vegetable and medicinal plant (Chinese lettuce, celtuce or “wosun”). Also in Kraków and surrounding area, local cultivars of the plant were once popular as a vegetable (gła˛biki krakowskie). Secondary metabolites synthesized by the plant have not been examined in detail. The only study on sesquiterpenoids from the stalks of a Chinese cultivar of the vegetable (purchased on local market) was published by Han et al. (2010). Sesquiterpene lactones produced by Lactuca ssp. display a vast array of biological activities (Wesołowska et al., 2006; Chadwick et al., 2013) and are deposited in aerial parts as well as in roots of lettuce plants (Zidorn, 2009). The roots, which are not edible, may provide a rich source of the active compounds. Ishihara et al. (1987), from a water extract of L. sativa fresh roots, isolated three sesquiterpene lactone glycosides: lactuside A, lactuside C and macrocliniside A. Our previous comparative HPLC study of eleven Lactuca species, based on distributional data of eight sesquiterpene lactones, revealed that roots of Lactuca sativa L. (cv. British Hilde) contained 8-deoxylactucin, jacquinelin, crepidiaside B, lactucin, 11b,13-dihydrolactucin, lactucopicrin and lactuside A. Moreover, we proved that roots of both cultivated and wild lettuce species are
* Corresponding author. E-mail address: [email protected] (K. Michalska).
richer in sesquiterpenes than aerial parts of the plants (Michalska et al., 2009). Thus, we decided to examine roots of asparagus lettuce cv. “Grüner Stern” in order to assess their usability as a source of sesquiterpene lactones. 2. Results and discussion The present study deals with isolation and identification of 21 known sesquiterpene lactones (Fig. 1, 1–21), together with four phenolic compounds, from roots of L. sativa var. angustana cv. “Grüner Stern”. The known compounds: 11b,13-dihydrolactucin (1), 11b,13dihydrolactucopicrin (2), crepidiaside B (3), cichorioside B (4), lactucin (5), lactucopicrin (6), leucodin (7), 8-deacetylmatricarin8-O-sulfate (8), 11b-hydroxyleucodin-11-O-b-glucopyranoside (9), scorzoside (10), 11b,13-dihydrovernoflexuoside (11), ixerin F (12), macrocliniside A (13), lactuside C (14), 10b,14-dihydroxy-10 (14),11b(13)-tetrahydro-8,9-didehydro-3-deoxyzaluzanin C-10-Ob-glucopyranoside (15), 3b,14-dihydroxy-11b,13-dihydrocostunolide (16), sonchuside A (17), ixerin H (18), picriside B (19), lactulide B (20) lactuside A (21), coniferyl aldehyde, sinapyl aldehyde, eugenyl-4-O-b-glucopyranoside and 3,4,5-trimethoxybenzaldehyde were identified by direct comparison of their spectral data either with those of the reference compounds formerly isolated in our laboratory (Michalska et al., 2014, 2015; Michalska and Kisiel, 2003, 2010, 2013) or with those from the literature (Mahmoud et al., 1986).
http://dx.doi.org/10.1016/j.phytol.2016.10.023 1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
426
K. Michalska et al. / Phytochemistry Letters 20 (2017) 425–428 Table 1 1 H NMR (600.20 MHz) and
13
C NMR (150.94 MHz) data of compound 20 in CDCl3.
Position
dH (ppm), J (Hz)
dC (ppm) HMBC (H ! C)
1 2a 2b 3 4 5 6 7 8a 8b 9a 9b 10 11 12 13 14a 14b 15
5.55 dd (8.1, 8.1) 2.17 ma 2.29 ddd (12.5, 11.3, 8.1) 4.05 dd (11.3, 2.5) – 5.03 br d (9.9) 4.69 dd (9.9, 9.9) 1.58 mb 2.17 ma 1.50 dddd (12.5, 11.5, 5.0, 1.7) 2.17 ma 2.05 ddd (15.0, 15.0, 5.0) – 2.22 dq (12.7, 6.9) – 1.23 d (6.9) 4.03 d (12.7) 4.08 d (12.6) 1.88 d (1.3)
123.28 33.84
a b
76.27 138.09 123.70 80.01 49.14 26.29 23.70 142.07 41.65 178.58 12.92 66.09 10.92
C-2, C-9, C-14 C-1, C-3, C-4 C-1, C-3, C-4 C-1, C-2, C-15 – C-3, C-7, C-15 C-4, C-8 C-5, C-8, C-11 C-6, C-7, C-9, C-10, C-11 C-6, C-7, C-9, C-10, C-11 C-1, C-7, C-8, C-10 C-1, C-7, C-8, C-10, C-14 – C-7, C-8, C-13 – C-7, C-11, C-12 C-1, C-9, C-10 C-1, C-9, C-10 C-3, C-4, C-5
Signals overlapped. Obscured by the signal of H2O.
and 2D NMR measurements, including COSY, NOESY, HMQC and HMBC spectra (Table 1). Analysis of the 1H NMR and 13C NMR spectra indicated that the compound 20 is a melampolide with two double carbon-carbon bonds in the molecule. The position and alpha configuration of the proton at the carbon bearing the hydroxyl group were established on the basis of its signal observed in the 1H NMR spectrum at dH 4.05 (dd, J = 11.3, 2.5 Hz), supported by the COSY and NOESY spectra. Thus a hydroxyl group at C-3 had beta configuration. In the 1H NMR spectrum of 20 two-proton AB systems at dH 4.08 (d, J = 12.6 Hz) and dH 4.03 (d, J = 12.7 Hz) at C-14 could be observed. The presence of an 11b,13-dihydroderivative of a methylene lactone was deduced from the occurrence of a methyl doublet at dH 1.23 (d, J = 6.9 Hz) and a double quartet at dH 2.22 (dq, J = 12.7, 6.9 Hz). The coupling constant of 12.7 Hz indicated an 11bproton. The relative stereochemistry of 20 was determined on the basis of coupling constants of the proton signals, the chemical shifts of the carbon signals as well as the results of NOESY experiments, by making an assumption that H-7 is alpha. The configurations of H-5a, H-6b, H-7a and H-11b were confirmed based on the values of J5a,6b = 9.9 Hz, J6b,7a = 9.9 Hz and J7a,11b = 12.7 Hz, which were indicative of anti-diaxial relationships of the respective protons. More relevant results were NOESY
Fig. 1. Chemical structures of compounds 1–21 (Glc = b-glucopyranosyl, A = 4hydroxyphenylacetyl).
The guaianolide 11b-hydroxyleucodin-11-O-b-glucopyranoside (9) was first obtained from Taraxacum obovatum (Michalska and Kisiel, 2003) and this is the first reported occurrence of 9 in the genus Lactuca. This is also the first report describing the presence of lactulide B (20) in Lactuca spp. The compound was originally isolated from Notoseris rhombiformis and was erroneously identified as 3b,14-dihydroxy-11b,13-dihydrocostunolide, which was not in accordance with the given NMR data taken in CD3OD (Liao et al., 2002). Moreover, the spectral data given by the authors were not complete. We deduced the structure of 20 from its 1H NMR spectrum in CDCl3. The structural assignment was verified by mass Fig. 2. Key NOESY correlations for 20.
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cross peaks from H-7a to H-3a and H-5a, and from H-6b to H-8b, H-9b, H-11b and H-15. E configuration of the double bond C1-C10 was proved by correlation of protons at C-2 and C-9 in the NOESY spectrum (Fig. 2). ESI mass spectrum of the compound displayed an adduct ion peaks at m/z: 289.0 [M+Na]+ and 555.3 [2M+Na]+ suggesting a molecular formula C15H22O4. Glucoside of 20 (lactuside B) was formerly isolated from roots of Lactuca laciniata Makino (Nishimura et al., 1986). By analogy with lactuside A and its aglycone–lactulide A we named the isolated melampolide (20) lactulide B. The isolated guaianolides: lactucin (5), lactucopicrin (6) and their dihydroderivatives as well as the germacranolide lactuside A (21), are characteristic constituents of cultivated lettuce plants (Tamaki et al., 1995; Sessa et al., 2000; Michalska et al., 2009; AbuReidah et al., 2013). 3b,14-Dihydroxy-11b,13-dihydrocostunolide (16) was found in the aerial parts of L. sativa by Mahmoud et al. (1986). Cichorioside B (4) and 8-deacetylmatricarin-8-O-sulfate (8) have been reported recently (Han et al., 2010; Abu-Reidah et al., 2013; Mai and Glomb, 2016) as constituents of some commercial varieties of lettuce and chicory. According to Tamaki et al. (1995), commercial varieties of lettuce are often devoid of 8-deoxylactucin. This is in agreement with the present study, though we previously found 8-deoxylactucin in roots and aerial parts of L. sativa cv. British Hilde (Michalska et al., 2009). The overall levels of bioactive lactucin-like guaianolides, possessing an exomethylene function, which were found in the commercially grown cultivars of lettuce were usually significantly lower comparing to those in closely related wild species of the genus Lactuca (Tamaki et al., 1995; Michalska et al., 2009; Seo et al., 2009). In the present study isolation yields of lactucin (5) and lactucopicrin (6) were 12.3 mg g1 and 29.8 mg g1, respectively, calculated on the dry weight basis. Macrocliniside A was the only exomethylene-bearing sesquiterpene lactone isolated with relatively high yield (179 mg g1). Han et al. (2010) isolated and identified eight guaianolides and three eudesmanolides from the stalks of commercially available chinese cultivar of L. sativa var. anagustata. Only four of these compounds: 11b,13-dihydrolactucin (1), 11b,13-dihydrolactucopicrin (2), cichorioside B (4) and macrocliniside A (13, isolation yield achieved by Han et al. 2.28 mg g1) were isolated from roots of the plant examined in our laboratory. We did not found any eudesmanolides which were previously described from the Chinese material. However, we isolated and identified six germacranolides (16–21) which were not reported by Han et al. (2010). Compounds 9 and 20 were isolated for the first time from the plant of the genus Lactuca. Lactuside A (21) and 8deacetylmatricarin-8-O-sulfate (8) were among the major sesquiterpene lactones found in the examined roots (isolation yields 160 mg g1 and 210 mg g1, respectively, calculated on a dry weight basis). Our present study reveals diversity of sesquiterpenes produced by closely related cultivars of commercially grown lettuce and usefulness of lettuce by-products as a source of biologically active compounds.
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with H2O-MeOH mixtures at a flow rate of 3.0 ml min1. Column chromatography was carried out using Merck silica gel 60 (0.063– 0.2 mm). TLC was performed on Merck silica gel 60 (0.25 mm) plates. 3.2. Plant material Roots of L. sativa var. angustana cv. “Grüner Stern” were collected in July 2014 from flowering plants grown in the Garden of Medicinal Plants, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland, where a voucher specimen (03/2014) was deposited. Seeds of the plant were derived from the Botanical Garden of the University of Bonn, Germany. 3.3. Extraction and isolation The dried plant material (228 g) was powdered and exhaustively extracted with ethanol at room temperature with shaking. After removal of the solvent under reduced pressure, the residue (10 g) was passed through a silica gel (Merck, Art. 7754) column using mobile phase gradients of EtOAc in hexane (up to 100% EtOAc), followed by EtOAc-MeOH (up to 5% MeOH). Elution of the column with hexane-EtOAc (7: 3, v/v) and subsequent prep. TLC (hexane-EtOAc, 7: 3, 3: 2, 1: 1) afforded coniferyl aldehyde (3.5 mg), 3,4,5-trimethoxybenzaldehyde (3.7 mg), 7 (12.5 mg) and sinapyl aldehyde (4.1 mg). Fractions from hexane-EtOAc (1: 1) elution furnished 2 (3.4 mg) and 6 (6.8 mg). Further fractions eluted with hexane-EtOAc (1: 1) after separation by prep. TLC (CHCl3-MeOH, 9: 1) and semiprep. HPLC (H2O-MeOH, 7: 3, 3: 2, 1: 1) gave 1 (3.6 mg), 5 (2.8 mg) and 20 (1.9 mg). Fractions eluted with EtOAc, subsequently submitted to prep. TLC (CHCl3-MeOH, 9: 1 or 17: 3) and semiprep. HPLC (H2OMeOH, 3: 1 or 1: 1) yielded 20 (1.6 mg), 16 (1.7 mg), 9 (0.9 mg), 17 and eugenyl-4-O-b-glucopyranoside in a mixture (ca. 4: 1, 1.5 mg), 10 (1.1 mg), 11 and 19 in a mixture (ca. 1: 1.5, 1.6 mg), and 18 (1.2 mg). Less polar fractions eluted with EtOAc-MeOH (49: 1) which were further separated by prep. TLC (CHCl3-MeOH, 17: 3) and semiprep. HPLC (H2O-MeOH, 7: 3) gave 21 (36.8 mg), 14 (21.9 mg), 3 and 15 in a mixture (ca. 1: 5, 5.3 mg) and 15 (6.0 mg). More polar fractions eluted with EtOAc-MeOH (49: 1) and next subjected to prep. TLC (CHCl3-MeOH, 17: 3) gave 12 and 13 in a mixture (ca. 3: 4, 69.1 mg), 8 and 4 in a mixture (ca. 4: 1, 34.8 mg) and 4, 12 and 13 in a mixture (ca. 1: 1: 1, 4.1 mg). 3.4. Lactulide B (20) Colourless solid; ½a26 D 12.6 (c = 0.53, CHCl3); ESIMS (pos. + mode) m/z: 289.0 [M+Na] and 555.3 [2M+Na]+; 1H NMR and 13C NMR: Table 1.
Acknowledgement The financial support of the Ministry of Science and Higher Education, Poland, (statutory activity founding) is gratefully acknowledged.
3. Experimental References 3.1. General experimental procedures All NMR spectra were recorded on a Bruker AVANCE III 400 and a Bruker AVANCE III 600. ESI mass spectra was obtained in the positive ion mode using AmaZon ETD (Bruker-Daltonics). Optical rotation was determined on a PolAAr 31 polarimeter. Semiprep. RP HPLC was performed on a Waters instrument coupled to a dual wavelength UV/VIS detector operating at 210 and 260 nm, using Delta-Pak C-18 column (particle size 15 mm, 25 100 mm) eluted
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