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Phenolic metabolites from Hypericum kelleri Bald ., an endemic species of Crete (Greece)
Phytochemistry 146 (2018) 1e7
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Phenolic metabolites from Hypericum kelleri BALD., an endemic species of Crete (Greece) Angeliki Mathioudaki a, b, Ariola Berzesta a, b, Zacharias Kypriotakis c, Helen Skaltsa a, €rg Heilmann b, * Jo a
Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, 157 71, Athens, Greece €t Regensburg, Pharmaceutical Biology, Universita €tsstr. 31, D-93053, Regensburg, Germany Universita c Technological Education Institute, School of Agricultural Production, Lab. of Taxonomy and Management of Wild Flora, Stavromenos P.O.Box 140, Heraklion-Crete, 71110, Greece b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2017 Received in revised form 13 November 2017 Accepted 22 November 2017
Thirteen compounds were isolated from the aerial parts of Hypericum kelleri BALD., growing as an endemic on the island of Crete (Greece). These compounds comprise four previously unknown prenylated xanthones 1,2-dihydro-3,8-dihydroxy-6-methoxy-1,1,5-tri(3-methylbut-2-enyl)xanthen-2,9-dione (kellerine A), 1,2-dihydro-3,6,8-trihydroxy-1,1,5-tri(3-methylbut-2-enyl)xanthen-2,9-dione (kellerine B), 1,2dihydro-3,8-dihydroxy-6-methoxy-1,1-bi(3-methylbut-2-enyl)xanthen-2,9-dione (6-methylpatulone), (R/S)-1,3,5-trihydroxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3-enyl]-6methoxy-9H-xanthen-9-one ((200 R/S)-kellerine C) and the hitherto undescribed depsidone (R/S)-1,3,6trihydroxy-5-methoxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3-enyl]-11Нdibenzo[b,e] [1,4]dioxepin-9-one ((200 R/S)-creticine). As known compounds, brevipsidone D, 4-geranyl-2(20 -isobutyryl)-phloroglucinol, 4-geranyl-2-(20 -methylbutyryl)-phloroglucinol, I3, II8-biapigenin, quercetin, avicularin, pseudohypericin and neochlorogenic acid have been isolated. The structures were elucidated on the basis of their 1D, 2D NMR, CD and MS data. The study confirms the typical occurrence of xanthones in Hypericum section Oligostema (BOISS.) STEF., and is also the first report on the simultaneous isolation of acylphloroglucinols in this section. Furthermore the first evidence of depsidones in the genus Hypericum L. is reported. Cytotoxicity was investigated in HeLa cells for prenylated xanthones and the depsidones. Both triprenylated 1,2-dihydroxanthones (kellerine A and B) showed significant in vitro cytotoxicity with IC50 values of 2.5 ± 0.1 (kellerine A) and 5.9 ± 0.9 (kellerine B) mM, whereas other compounds were less cytotoxic (IC50 > 20 mM). © 2017 Published by Elsevier Ltd.
Dedicated to Prof. Dr. Otto Sticher on the occasion of his 80th birthday and as homage to his outstanding phytochemical and analytical work. Keywords: Hypericum kelleri Hypericaceae Xanthones Kellerines A-C Chemotaxonomy Depsidones Creticine
1. Introduction Hypericum L. is a large genus with pharmaceutically relevant representatives, which comprises ~470 species of trees, shrubs, and herbs οf worldwide distribution, but most species originate from temperate regions and tropical mountains (Crockett and Robson, 2011). Several of them have been the subject of chemical and pharmacological investigations and proved a rich source of acylphloroglucinols, xanthones and flavonoids (Ernst, 2003; Avato, 2005; Karioti and Bilia, 2010).
* Corresponding author. E-mail address: [email protected] (J. Heilmann). https://doi.org/10.1016/j.phytochem.2017.11.009 0031-9422/© 2017 Published by Elsevier Ltd.
The present study deals with the first phytochemical investigation of H. kelleri BALD., (Hypericaceae) belonging to the section Oligostema (BOISS.) STEF., a narrow endemic species in western Crete. The flora of the island includes 1825 native vascular plant species, 10.6% of which are single island endemic species, with a distribution range restricted to the main island of Crete (Trigas et al., 2013). The Cretan taxons of Hypericum share a similar habitus as dwarf shrubs and showed a preferred distribution in the low mountain range. Nevertheless, they exhibit different morphology with regards to glands and leaves and belong to different sections. Three species of the section Coridium namely H. empetrifolium (Schmidt et al., 2012a and b), H. amblycalyx COUST. & GAND. (Winkelmann et al., 2003) and H. jovis GREUT. (Athanasas et al., 2004), have been phytochemically investigated, all showing
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prenylated mono, di- and tricyclic acylphloroglucinols as typical specialized metabolites. Most of these generates from an activated isobutyric or 2-methylbutyric acid as biosynthetic starter molecule. In contrast, H. aciferum (GREUT) N. ROBSON (sect. Adenotrias), H. trichocaulon BOISS. & HELDR. (sect. Drosocarpium) and H. kelleri (sect. Oligostema) have not been investigated up to now.
2. Results and discussion The cyclohexane extract of the aerial parts of Hypericum kelleri BALD. was fractionated by centrifugal partition chromatography followed by silica column chromatography and semi-preparative HPLC and yielded previously undescribed xanthones (Fig. 1) 1,2dihydro-3,8-dihydroxy-6-methoxy-1,1,5-tri(3-methylbut-2-enyl) xanthen-2,9-dione (kellerine A, 1), 1,2-dihydro-3,6,8-trihydroxy1,1,5-tri(3-methylbut-2-enyl)xanthen-2,9-dione (kellerine B, 2), 1,2-dihydro-3,8-dihydroxy-6-methoxy-1,1-bi(3-methylbut-2-enyl) xanthen-2,9-dione (6-methyl-patulone, 3), (R/S)-1,3,5-trihydroxy2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-
methylbut-3-enyl]-6-methoxy-9H-xanthen-9-one ((200 R/S)-kellerine C, 4), as well as the unknown depsidone (R/S)-1,3,6-trihydroxy5-methoxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2enyl)-3-methylbut-3-enyl]-11Н-dibenzo[b,e] [1,4]dioxepin-9-one ((200 R/S)-creticine, 5) and known brevipsidone D (6) (Ngoupayo et al., 2008). The dichloromethane extract of the same plant material was fractionated by flash chromatography, CPC (Centrifugal partition chromatography) and RP18-HPLC and yielded 4-geranyl2-(20 -isobutyryl)-phloroglucinol (7) (Crockett et al., 2008; Rios and Delgado, 1992), 4-geranyl-2-(20 -methylbutyryl)-phloroglucinol (8) (Crockett et al., 2008; Rios and Delgado, 1992). Finally, the methanol extract was fractionated by liquid-liquid extraction and column chromatography on Sephadex® LH-20 and afforded quercetin (9) (Tatsis et al., 2007; Chang et al., 2009), avicularin (10) (Chang et al., 2009), I3,II8-diapigenin (11) (Tatsis et al., 2007), pseudohypericin (12) (Tatsis et al., 2009; Karioti et al., 2009) and neochlorogenic acid (13) (Hyun et al., 2010). The structures of the compounds were established on the basis of 1D and 2D NMR as well as CD and HRMS techniques. Compounds
Fig. 1. Structures of compounds 1e5.
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1e4 were isolated as yellow oily residues and showed similar NMR features with scaffold characteristic signals referring to xanthone derivatives. Compound 1 had a molecular formula of C29H34O6 based on HRESIMS m/z 477.2289 [M-H]-. The 1H NMR spectrum of 1 showed the presence of a hydrogen-bonded hydroxyl proton at dH 13.23 (1H, s, 8-ΟН), two aromatic protons at dH 6.51 (1H, s, Н-4) and 6.39 (1H, s, Н-7), one methoxy-group at dH 3.89 (3H, s, 6-OCH3) and one hydroxyl proton at dH 7.00 (1H, s, 3-OH). In addition, the 1H NMR spectrum revealed signals at dН 4.64 (2Н, t, J ¼ 7.6 Hz, H-20 and H-200 ), 3.43 (2H, dd, J ¼ 7.6, 13.7 Hz, H-1a0 and H-1a00 ), 2.80 (2H, dd, J ¼ 7.6, 13.7 Hz, H-1b0 and H-1b00 ), 1.48 (6Н, s, H-40 and H-400 ) and 1.49 (6H, s, H-50 and H-500 ) characteristic of the presence of gem di (3-methylbut-2-enyl)-side chains (Azebaze et al., 2006). Furthermore, the rest of the signals at dН 3.43 (2H, d, J ¼ 7.2 Hz, H-1000 ), 5.21 (1H, t, J ¼ 7.2 Hz, H-2000 ), 1.83 (3Н, s, Н-4000 ) and 1.69 (3H, s, Н-5000 ) revealed the presence of one more 3-methylbut-2-enyl lateral chain connected. These data suggested that 1 is a xanthone derivative having three prenyl side chains. 1H,1H COSY spectrum confirms the proton correlations within the prenyl sidechains and 1 13 H, C HSQC enable the respective assignment of the methines, methylenes and methyl groups. In the 1H,13C HMBC spectrum, the following key long-range correlations were observed: OH-3 only with C-4 (dC 108.9) while H-4 correlated with C-2 (dC 201.3), C-3 (dC 151.7), C-4a (dC 159.2), C-9 (dC 179.8), C-9a (dC 116.2); 8-ΟН with C-6 (dC 162.6), C-7 (dC 95.3), C- 8 (dC 161.1), C-8a (dC 104.7); 6-OCH3 with C-6. The long-range correlations of the CH2-10 and 100 protons with C-1 (dC 55.9) and C-2 revealed that the gem 3-methylbut-2-enyl lateral chains are located at C-1. Furthermore, the HMBC cross peaks between CH2-1000 and C-4b (dC 153.3), C-5 (dC 107.2) and C-6 revealed that the third 3-methylbut-2-enyl side chain is attached to C-5 (dC 107.2). Methoxylation at C-6 was verified by 1H,1H ROESY correlations of methyl protons with H-7 and the observed hydrogen bond of hydroxyl group at C-8. On the basis of these data the hitherto unknown structure of 1 was determined as 1,2-dihydro3,8-dihydroxy-6-methoxy-1,1,5-tri(3-methylbutyl-2-enyl) xanthen-2,9-dione and named kellerine A. The 1H and 13C NMR spectroscopic data of compound 2, C28H32O6, were very similar to those of 1 except for the substituent at C-6. The 1H NMR data showed the absence of the signal at dН 3.89 (3H, s) and the presence of one hydroxyl group at dН 5.95 (1H, s, 6OH); HMBC cross peaks with C-5 (dC 104.7), C-6 (dC 160.7) and C-7 (dC 99.6) confirmed the site of attachment. 1H,1H COSY and 1H,13C HSQC spectra also matched the 2D NMR analysis of 1. Thus, compound 2 was also isolated for the first time and determined as 1,2dihydro-3,6,8-trihydroxy-1,1,5-tri(3-methylbut-2-enyl)xanthen2,9-dione and was named kellerine B. The main difference at the 1H and 13C NMR spectroscopic data of compound 3, C24H26O6, compared to 1, is the absence of the signals corresponding at the 3-methylbutyl-2-enyl side chain of C-5. Instead, in the 1H NMR spectrum, one more methine at dН 6.39 (H5) appeared as doublet with meta coupling with H-7 (dН 6.36, J ¼ 2.2), while in 13C NMR, C-5 (dC 92.2) resonated upfield compared with 1 and 2. This was confirmed by an additional crosspeak (dН 6.36/6.39) in 1H,1H COSY and an additional crosspeak for dH/C 6.39/ 92.2 in 1H,13C HSQC spectrum. Thus, previously undescribed compound 3 was determined as 1,2-dihydro-3,8-dihydroxy-6methoxy-1,1-bi (3-methylbut-2-enyl)xanthen-2,9-dione and was named 6-methyl-patulone based on the structural similarities with the previously isolated patulone (Ishiguro et al., 1997). Compound 4, obtained as a yellow oily residue, showed a molecular formula of C29H34O6 on the basis of HRESIMS m/z 477.2286 [M-H]-. The 1H NMR spectrum showed the characteristic signals of two ortho-aromatic protons at dН 6.97 (1Н, d, J ¼ 8.8 Hz, H-7) and 7.81 (1H, d, J ¼ 8.8 Hz, H-8), one hydrogen-bonded hydroxyl proton at dH 13.31 (1H, s), two phenolic hydroxyl groups at dH 6.28 (1H, s,
3
3-OH) and 5.63 (1H, s, 5-OH), one methoxy group at dH 4.04 (3H, s), one prenyl group [dH 3.48 (2H, d, J ¼ 7.2 Hz, H-10 ); dH 5.27 (1H, t, J ¼ 7.2 Hz, H-20 ); dH 1.86 (3H, s, H-40 ) and dH 1.78 (3H, s, H-50 )], and a 2-(3-methylbut-2-enyl)-3-methylbut-3-enyl-group [dH 2.91 (2H, d, J ¼ 7.2 Hz, H-100 ); dH 2.51 (1H, quint, J ¼ 7.2 Hz, H-200 ); dH 2.19/2.18 (2H, t, J ¼ 7.1 Hz, H-1000 ); dH 5.13 (1H, t, J ¼ 7.1 Hz, H-2000 ); dH 1.58 (3H, s, H-4000 ); dH 1.68 (3H, s, H-5000 ); dH 4.64/4.68 (2H, brs, H-400 ) and dH 1.75 (3H, s, H-500 )]. The 13C spectra displayed signals of 29 carbons including 14 quaternary, 5 methine, 4 methylene, and 6 methyl carbons, which could be well assigned to a substituted xanthone skeleton. The seven substituents on the xanthone skeleton were determined on the basis of 1H,13C HMBC, 1H,1H COSY and 1H,13C HSQC spectral analysis. In the HMBC spectrum, the hydrogenbonded hydroxyl proton correlated with C-1 (dC 158.4); C-2 (dC 108.4) and C-9a (dC 102.7). The prenyl group was located at C-2, since its typical methylene protons at dH 3.48 showed HMBC correlations with C-1 (dC 158.4); C-2 (dC 108.4); C-3 (dC 160.7); C-20 (dC 121.3) and C-30 (dC 136.0). The presence of a 2-(3-methylbut-2enyl)-3-methylbut-3-enyl-group was elucidated by 1H,1H COSY and 1H,13C HSQC correlations. It is located at C-4, since the methylene protons at dH 2.91 showed HMBC correlations with C-4 (dC 106.1); C-4a (dC 149.0) and C-3 (dC 160.7) and the methylene protons at dH 2.18/2.19 showed HMBC correlations with C-4a (dC 149.0, J5). The protons of the methoxy group correlated with C-6 (dC 151.1). The ortho-aromatic protons at dH 6.97 and 7.81 were assigned as H7 and H-8, respectively, according to the coupling between the proton at dH 7.81 and the carbonyl carbon C-9 (dC 180.9). The two phenolic hydroxyl groups at dH 6.28 (1H, s) and 5.63 (1H, s) correlated with C-3 (dC 160.7) and C-5 (dC 133.4), respectively. To determine the absolute stereochemistry for C-200 , a CD-spectrum was recorded. In comparison to experimental CD data gained for the very similar xanthones monogxanthone A and B (Xu et al., 2016) with the concerning C being R-configurated, kellerine C must be racemic due to lacking Cotton effects. Thus, the structure of 4 was identified as the previously unknown (R/S)-1,3,5-trihydroxy2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3methylbut-3-enyl]-6-methoxy-9H-xanthen-9-one and was named (200 R/S)-kellerine C. The 6-desmethylderivative of kellerine C was previously isolated from H. monogynum L. (syn. H. chinense, not accepted) by Tanaka et al. (2010) and Xu et al. (2016) and named biyouxanthone D. Compound 5, obtained as a yellow oily residue, showed a molecular formula of C29H34O7 based on HRESIMS m/z 493.2236 [MH]-. The proton signals in the 1H NMR spectrum were quite similar to the signals of compound 4 showing again the characteristic signals of two ortho-aromatic protons at dН 6.75 (1Н, d, J ¼ 9.1 Hz, H-7) and 6.93 (1H, d, J ¼ 9.1 Hz, H-8), two phenolic hydroxyl groups at dH 6.27 (1H, s, 3-OH) and 5.53 (1H, s, 6-OH), one methoxy group at dH 3.99 (3H, s), one prenyl group [dH 3.40 (2H, d, J ¼ 7.1 Hz, H-10 ); dH 5.17 (1H, t, J ¼ 7.1 Hz, H-20 ); dH 1.81 (3H, s, H-40 ) and dH 1.66 (3H, s, H-50 )], and a 2-(3-methylbut-2-enyl)-3-methylbut-3-enyl-group [dH 3.00 (2H, d, J ¼ 7.4 Hz, H-100 ); dH 2.51 (1H, quint, J ¼ 7.4 Hz, H200 ); dH 2.14 (2H, t, J ¼ 6.9 Hz, H-1000 ); dH 5.04 (1H, t, J ¼ 6.9 Hz, H-2000 ); dH 1.56 (3H,s, H-4000 ); dH 1.66 (3H, s, H-5000 ); dH 4.78/4.71 (2H, brs, H400 ) and dH 1.75 (3H, s, H-500 )]. 1H,1H COSY and 1H,13C HSQC spectra also showed matching correlations. The only main difference was regarding the 1H shift of the hydrogen-bonded hydroxyl proton, which resonated upfield shifted at dH 11.08 ppm together with the downfield shift of the aromatic protons H-7 and H-8. In addition, dC values at the 13C spectra of compounds 4 and 5 were quite similar, except of the C-8a, which was de-shielded to dC 138.7 ppm. The molecular formula of compound 5 confirms that an additional oxygen causes this de-shielding. Accordingly, C-9 of 5 is not an unsaturated keto functionality, but now an ester showing an upfield shift from dC 180.9 in 4 to dC 168.0 ppm in 5. Again, lacking Cotton
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effects in ECD spectrum and comparison to experimental and calculated CD data (Xu et al., 2016) showed the presence of an racemate. Thus, compound 5 was identified as the previously undescribed depsidone derivative (R/S)-(1,3,6-trihydroxy-5methoxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)3-methylbut-3-enyl]-11Н-dibenzo[b,e][1,4]dioxepin-9-one and was named (200 R/S)-creticine. H. kelleri BALD. belongs to the section Oligostema (BOISS.) Stef., together with H. andjerinum Font Quer & Pau, H. australe TEN (¼H. humifusum subsp. australe (TEN.) ROUY & FOUCAUD ¼ H. linarifolium BERTOL.), H. humifusum L., H. linearifolium WILLD. (¼ H. linarifolium VAHL ¼ H. humifusum subsp. linarifolium (Vahl) ROUY & FOUCAUD) and H. repens L. (¼ H. tenellum E. D. CLARKE) based on similarities in floral and vegetative morphology (Robson, 2010; Nürk and Crockett, 2011). Few phytochemical studies have been carried out for most of these species revealing the presence of xanthones (i.e. mangiferin/isomangiferin), flavonoids, phenolic acids and naphthodianthrones. This is the first phytochemical study on H. kelleri. Concerning the presence of xanthones, our results are in agreement to previous studies in Oligostema; despite the fact that they occur as aglycones and not as C-glycosides in H. kelleri, it seems to be a typical class of specialized metabolites in this section. It is noteworthy that kellerines are oxidized (and methoxylated) derivatives of tetrahydroxy xanthones and therefore structurally closely related to mangiferin, so far, found in H. humifusum (Kitanov and Nedialkov, 1998; Battistini, 2013), H. humifusum subsp. australe (Rouis et al., 2013) and isomangiferin in H. linearifolium (Seabra and Alves, 1989). Moreover, 4-geranyl-2(20 -isobutyryl)-phloroglucinol and 4-geranyl-2-(20 -methylbutyryl)-phloroglucinol found in H. kelleri are the first isolated acylphloroglucinols from Oligostema, to the best of our knowledge. Furthermore, this study is the first evidence of depsidones in the genus Hypericum L. and just the second one into the Plantae kingdom, while they have been isolated only from the genus Garcinia till today (Permanaa et al., 2005; Ito et al., 2001; Xu et al., 2000). Among the different groups of specialized metabolites present in Hypericum L. the polyphenolic metabolites pseudohypericin, hypericin as naphtodianthrons, quercetin, isoquercitrin, rutin as flavonoids and the phenylpropanoids chlorogenic and neochlorogenic acids have been also often investigated. Previous studies in taxa belonging to Oligostema revealed their presence as follows: hypericin has been found in H. humifusum (Umek et al., 1999; Farinha et al., 2002), H. linearifolium (Farinha et al., 2002) and H. repens (Makovetska, 2000); pseudohypericin in the latter two species (Makovetska, 2000; Farinha et al., 2002); quercetin and hyperoside in H. humifusum (Lebreton and Bouchez, 1967; Umek et al., 1999; Farinha et al., 2002), as well as quercitrin, isoquercetrin, amentoflavone, I3,II8-biapigenin in H. humifusum (Umek et al., 1999; Farinha et al., 2002), while in H. linearifolium have been found all the previous quoted flavonoids, except the last one, plus rutin (Farinha et al., 2002). Moreover, chlorogenic acid has been found in H. humifusum, H. linearifolium (Rouis et al., 2013); its methyl ester, neochlorogenic acid and 3-O-p-coumaroyl quinic acid in H. humifusum ssp. australe (Rouis et al., 2013; Battistini, 2013). Although these compounds are not considered as chemotaxonomic markers, they are related to the pharmacological effects of the plants (Crockett and Robson, 2011). Consequently, we also investigated the methanol fractions and were able to isolate the naphthodianthrone pseudohypericin (12), the flavonoids quercetin (9), avicularin (10) and I3,II8-biapigenin (11), as well as neochlorogenic acid (13). Extensive TLC analyses of enriched Sephadex-LH20 fractions confirm the lack of traceable amounts of hypericin, kaempferol, isoquercitrin, hyperoside, rutin and amentoflavone. It is worth mentioning that during the whole isolation course, main
constituents, as well as minor ones were continuously monitored and traced down using an NMR metabolomic strategy, which permitted in detail characterization of extracts and sub fractions thereof. Cytotoxicity was evaluated for hitherto unknown compounds (1e5) and brevipsidone D in HeLa cells. Only the 1,2dihydroxanthones substituted by three prenyl groups (1 and 2) showed significant in vitro cytotoxicity with IC50 values of 2.5 ± 0.1 (1) and 5.9 ± 0.9 (2) mM, whereas other compounds were less cytotoxic (IC50 > 20 mM). 3. Experimental section 3.1. General experimental procedures Optical rotations were recorded using a UniPol L1000 polarimeter (Schmidt þ Haensch) and a Perkin-Elmer 341 Polarimeter. UV spectra were obtained in MeOH on a Cary 50 Scan spectrophotometer (Varian) according to Mabry et al. (1970). CD-data were gained in MeOH on a J-715 spectropolarimeter (JASCO) at 22 C from 200 to 400 nm with the compounds concentrated with 200 mmol/L. 1H, 13C and 2D NMR spectra were recorded in CDCl3 and CD3OD on Bruker Avance III 600 equipped with a 5 mm TCI CryoProbe, Bruker DRX 400, Bruker Avance III HD 400 and Bruker AC 200 instruments at 295 K. Chemical shifts are given in ppm (d) and were referenced to the solvent signals at 7.24/3.31 and 77.0/ 49.0 ppm for 1H and 13C NMR, respectively. 1H,1H COSY, 1H,13C HSQC, 1H,13C HMBC, and 1H,1H ROESY (mixing time 950 ms) were performed using standard Bruker microprograms. High resolution mass spectra were measured on a Finnigan MAT SSQ 710 A at 70 eV (HREIMS, positive mode) or recorded on an Agilent 6540 UHD (HRESIMS, positive and negative mode). The solvents used were of spectroscopical grade (Merck). Column chromatography: silica gel (Geduran® Si 60 for column chromatography; Merck), gradient elution with the solvent mixtures indicated in each case. Semipreparative HPLC separations were performed on a Varian ProStar 210 Solvent Delivery Module equipped with a Varian ProStar 335 Photodiode Array Detector using as RP-18 HPLC column an Eclipse XDB-C18 (5 mm, 9.4 250 mm, Agilent, USA). All solvents used were of HPLC grade (Merck). Centrifugal partition chromatography was performed on an Armen Instrument Spot CPC (Alpha Crom, Laboratory equipment), with a 515 HPLC-pump (Waters, USA) and the fractions were collected automatically by a LKM Bromma 2211 Superrac instrument (SE). All solvents used were of p.a. grade (Merck). Silica gel 60 F254 precoated aluminium sheets and silica gel 60 RP-18 F254s precoated sheets (both from Merck) were used for TLC. Fractionation was always monitored by TLC silica gel 60 F-254, (Merck, Art. 5554) and silica gel 60 RP-18 F254s (Merck, Art. 15685) plates with visualization under UV (254 and 365 nm) and spraying with anisaldehyde-sulfuric acid reagent (anisaldehyde, 2 mL; concentrated H2SO4, 10 mL; HOAc, 16 mL; MeOH, 170 mL), macrogol 400 reagent (5 g PEG; 100 mL MeOH) and with Neu’ s reagent for phenolics (Neu, 1957). 3.2. Plant material Aerial parts of H. kelleri BALD. ((Hypericaceae) were collected on the whole plateau of Omalos (35 200 3700 N, 23 540 100 ; ~1040e1250 m), in the western part of Crete in June 2013 (regional district Chania). The plant was identified by Dr. Z. Kypriotakis. A voucher specimen is deposited at the Herbarium of Technological Education Institute, School of Agricultural Production, Lab. of Taxonomy and Management of Wild Flora (Heraklion-Crete, Greece) with the identification number 15925/26-6-2013.
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5
Table 1 1 Н NMR of compounds 1e3 (CDCl3, 600 MHz, 295 K).Chemical shifts are given in ppm and J in Hz. Position
1
HMBC
2
HMBC
3
HMBC
4 5 7 1a0 , 1a00 1b0 , 1b00 20 , 200 40 , 400 50 , 500 1000 2000 4000 5000 3-OH 6-ΟR 8-ΟН
6.51 s e 6.39 s 3.43 dd (7.6, 13.7) 2.80 dd (7.6, 13.7) 4.64 t (7.6) 1.48 s 1.49 s 3.43 d (7.2) 5.21 t (7.2) 1.83 s 1.69 s 7.00 s 3.89 s 13.23 s
2, 3, 4a, 9, 9a e 5, 6, 8a, 8 1, 2, 9a, 10 , 20 , 30 ,100 , 200 , 300
6.51 s e 6.30 s 3.44 dd (7.6, 13.7) 2.80 dd (7.6, 13.7) 4.64 t (7.6) 1.48 s 1.49s 3.52 d (7.2) 5.30 t (7.2) 1.87 s 1.78 s 6.95 s 5.95 s 13.14 s
2, 3, 4a, 9, 9a e 8a, 8 1, 2, 10 , 20 , 30 , 100 , 200 , 300
6.49 s 6.39 d (2.2) 6.36 d (2.2) 3.44 dd (7.7, 13.8) 2.79 dd (7.7, 13.8) 4.63 t (7.6) 1.48 s 1.48 s e e e e 6.96 s 3.87 s 13.15 s
2, 3, 4a, 9, 9a 4b, 6, 7, 8, 8a 5, 6, 8, 8a 1, 2, 10 , 20 , 30 , 100 , 200 , 300
10 , 40 , 50 , 100 , 400 , 500 1, 10 , 20 , 30 , 50 , 100 , 200 , 300 , 500 1, 10 , 20 , 30 ,40 , 100 , 200 , 300 ,400 4b, 5, 6, 2000 , 3000 1000 , 4000 , 5000 2000 , 3000 , 5000 2000 , 3000 , 4000 4 6 6, 7, 8, 8a
10 , 40 , 50 , 100 , 400 , 500 20 , 30 , 50 ,200 , 300 , 500 20 , 30 ,40 , 200 , 300 ,400 4b, 5, 6, 2000 , 3000 1000 , 4000 , 5000 2000 , 3000 , 5000 2000 , 3000 , 4000 2, 3, 4, 4a 5, 6, 7 7, 8, 8a
10 , 40 , 50 , 100 , 400 , 500 1, 20 , 30 , 50 , 200 , 300 , 500 1, 20 , 30 , 40 , 200 , 300 ,400 e e e e 2, 3, 4 6 7, 8, 8a
1, 3: R¼CH3, 2: R¼H.
3.3. Cell culture and determination of cytotoxicity HeLa cells (ATCC CCL17) were cultured at 37 C in a humidified incubator with 5% CO2. Culture medium was MEM (Biochrom AG) supplemented with 10% FCS and 2 mM L-glutamine. Cytotoxicity was evaluated in an MTT assay assessing the metabolic activity of NADPH-dependent cellular oxidoreductase enzymes capable of reducing MTT to its insoluble formazan as described by Heilmann et al. (2001). Tests were performed in duplicates and all experiments repeated three times (n ¼ 8). IC50 values were calculated from eight different concentrations and data are reported as mean ± SD. Maximal observed (absolute) standard deviation was about 15%. Positive control measurements were performed with xanthohumol. 3.4. Extraction and isolation Air-dried and powdered aerial parts of Hypericum kelleri BALD. (258.1 g) were extracted by maceration with cyclohexane, DCM, MeOH, and 5:1 (v/v) MeOH-H2O mixture, successively. The cyclohexane-soluble material (2.64 g) was defatted using MeOH at 0 C and the supernatant layer resulted after removal of solvent under vacuum a residue (2.17 g), which was subjected to centrifugal partition chromatography using a solvent system of MeOH-heptane (1:1) (flow rate 10 mL/min; rotation: 800 rpm; ASC) and resulted 434 fractions. Based on TLC similarities, identical fractions were combined to give a total of 11 fractions (KEC.1KEC.11). Part of KEC.5 (0.010 g) subjected to further purification by RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) using a H2OACN gradient (60% ACN 0e5 min; 60% / 97% ACN 5e20 min; 97% ACN 20e22 min; 97% / 80% ACN 22e23 min; 80% ACN 23e33 min; 80% / 60% ACN 33e34 min; 60% ACN 34e44 min) yielded compound 1 (5.1 mg, tR 28.19). Applying a H2O-ACN gradient (60% ACN 0e5 min; 60% / 97% ACN 5e25 min; 97% ACN 25e30 min; 97% / 60% ACN 30e31 min; 60% ACN 31e41 min) to fraction KEC.6 (8.0 mg) subjected to RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) yielded compound 3 (1.5 mg, tR 21.71). Fraction KEC.9 (17.5 mg) was subjected to RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) using the same gradient system as before to fraction KEC.6 yielded compound 2 (9.1 mg, tR 20.85). Part of fraction KEC.10 (88.1 mg) was applied to column chromatography over silica gel (50.5 1.5 cm) eluted with mixtures of increasing polarity of cyclohexane-EtOAc (flow rate 1.5 mL/min) and yielded 432 fractions (z1.5 mL each), which were combined based on TLC similarities to 11 fractions (KEC.10.1-KEC.10.11) one of which constitutes compound 5 (2.8 mg; fraction KEC.10.2). KEC.10.4 (11.6 mg)
subjected to further purification by RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) using a H2O-ACN gradient (60% ACN 0e5 min; 60% / 97% ACN 5e25 min; 97% ACN 25e30 min; 97% / 60% ACN 30e31 min; 60% ACN 31e41 min) and yielded compound 6 (1.1 mg; tR 21.5) and compound 4 (0.9 mg; tR 26.91). The dichloromethane extract (5.75 g) was defatted by CC through Diaion LH-20 and eluted with MeOH 90%, MeOH 100% and DCM to yield three fractions (DC 1e3). DC-1 was further fractionated by flash chromatography (Geduran® 60) eluted with mixtures of increasing polarity of hexane-EtOAc (flow rate 50 mL/min) and yielded 373 fractions (z23 mL each), which were combined based on TLC similarities to 12 fractions (F1-F12). Fraction F10 was subjected to CPC using hexane-EtOAc-MeOH-H2O 7:3:7:3 (flow rate 5 mL/min; rotation: 1200 rpm) and afforded 312 fractions combined, based on TLC similarities, to 7 sub-groups (F10.1 and F10.7). Fraction F10.2 was subjected on flash chromatography (Lichoprep® RP-18; 26 mm 12 cm) using a H2O-MeOH gradient (50% MeOH 0e2 min; 50% / 100% MeOH 2e60 min; 100% MeOH 60e105 min) and yielded 7 sub-fractions (F10.2.1- F10.2.7). Fraction F10.2.3 was
Table 2 13 C NMR of compounds 1e3 (CDCl3, 150 MHzat 295 K). Chemical shifts are given in ppm. Position
1
2
3
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 /100 20 /200 30 /300 40 /400 50 /500 1000 2000 3000 4000 5000 6-ΟR
55.9 201.3 151.7 108.9 159.2 153.3 107.2 162.6 95.3 161.1 104.7 179.8 116.2 37.9 117.8 135.2 17.9 25.7 21.6 121.9 131.9 17.9 25.8 56.0
55.9 201.4 151.8 108.7 154.0 153.8 104.7 160.7 99.6 160.5 105.1 179.7 116.4 37.9 117.7 135.3 17.9 25.7 21.7 121.2 134.8 18.0 25.8 e
56.1 201.2 151.7 108.7 158.8 156.0 92.2 165.4 98.2 162.6 105.1 179.2 116.9 38.2 117.6 135.5 18.0 25.6
1, 3: R¼CH3, 2: R¼H.
e e e e 55.9
6
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
Table 3 1 Н and 13C NMR of compounds 4 and 5 (CDCl3, 600 MHz for 1H, 150 MHz for a
C
mult
4
5
4
dC
dC
dH
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 20 30 40 50 100 200
C C C C C C C C CH CH C C C CH2 CH C CH3 CH3 CH2 CH
158.4 108.4 160.7 106.1 149.0 144.4 133.4 151.1 107.3 117.0 115.6 180.9 102.7 21.6 121.3 136.0 18.0 25.9 27.6 47.3
160.1 111.0 160.9 112.3 158.6 143.8 138.5 147.6 111.5 116.4 138.7 168.0 98.5 22.3 120.8 136.3 17.9 25.7 27.8 46.9
300 400 500 1000 2000 3000 4000 5000 1-OH 3-OH 5-OH 6-ΟН 5-OCH3 6-OCH3
C CH2 CH3 CH2 CH C CH3 CH3 e e e e CH3 CH3
153.4 111.0 19.3 31.7 123.1 132.5 17.9 25.8 e e e e
148.3 111.4 19.7 30.8 122.7 132.5 18.0 25.8 e e e e 62.9
a
56.6
13
C at 295 K). HMBC of 4
5
HMBC of 5
dH
6.97 d (8.8) 7.81 d (8.8)
4b, 5, 6, 8a 4b, 5, 6, 9
6.75 d (9.1) 6.93 d (9.1)
4b, 5, 6 4b, 5, 6
3.48 d (7.2) 5.27 t (7.2)
1, 2, 3, 20 , 30 1 0 , 40 , 50
3.40 d (7.1) 5.17 t (7.1)
2, 3, 20 , 30 10 , 40 , 50
1.86 s 1.78 s 2.91 d (7.2) 2.51 quint (7.2)
20 , 30 , 50 20 , 30 ,40 3, 4, 4a, 200 , 300 , 1000 4, 4a, 100 , 400 , 500 , 1000 , 2000
1.81 s 1.66 s 3.00 d (7.4) 2.51quint (7.4)
20 , 30 , 50 20 , 30 ,40 3, 4a, 200 , 300 , 400 , 1000 100 , 300 , 400 , 500 , 1000 , 2000
4.68, 4.64 br s 1.75 s 2.19, 2.18 t (7.1) 5.13 t (7.1)
200 , 500 4a, 200 , 400 4a, 100 , 200 , 2000 , 3000 200 , 1000 , 4000 , 5000
4.78, 4.71 br s 1.75 s 2.14 t (6.9) 5.04 t (6.9)
200 ,500 200 , 300 , 400 , 100 , 200 , 300 , 2000 , 3000 200 , 1000 , 4000 , 5000
1.58 s 1.68 s 13.31 s 6.28 s 5.63 s
2000 , 3000 , 5000 2000 , 3000 , 4000 1, 2, 9a 2, 3, 4 4b, 5, 6
1.56 s 1.66 s 11.08 s 6.27 s e 5.53 s 3.99 s
2000 ,3000 ,5000 2000 ,3000 ,4000 1, 2, 9a 2, 3, 4 e 5, 6,7 5
4.04 s
6
Determined by HSQC and HMBC correlations.
subjected to further purification by RP18-HPLC (Agilent XDB-C18 PrepHT; flow rate 1 mL/min) using a MeOH (50%)-2-propanol gradient (35% 2-propanol 0e18 min; 35% / 50% 2-propanol 18e21 min) and yielded compounds 7 (7.75 mg, tR 0.28) and 8 (29.23 mg, tR 0.29). The MeOH extract (29.16 g) was diluted to water and extracted with EtOAc and butanol, successively. Based on the TLC results, the EtOAc extract (8.26 g) was selected for further fractionation. Part of this extract (734.1 mg) was purified over column chromatography on Sephadex LH-20 (30 4 cm) eluted with MeOH 100% (flow rate 0.5 mL/min) and yielded 210 fractions (z10 mL each), which were combined based on TLC similarities to 14 fractions (KEM.A.Se1KEM.A.Se14), three of which constituted compounds 10 (32.4 mg, fraction KEM.A.Se7), 9 (17.6 mg, fraction KEM.A.Se12). 11 (6.6 mg, fraction KEM.A.Se13) and 12 (KEM.A.Se14, 7.1 mg). Furthermore KEM.A.Se1 (155.2 mg) was applied to column chromatography over silica gel (8.5 2.5 cm), eluted with mixtures of increasing polarity of DCM-MeOH-H2O (flow rate 2 mL/min) and yielded compound 13 (9.4 mg, DCM-MeOH-H2O 76:24:2.4). 3.4.1. Kellerine A (1,2-dihydro-3,8-dihydroxy-6-methoxy-1,1,5tri(3-methylbut-2-enyl)xanthen-2,9-dione) (1) Yellowish Oil; UV (MeOH) lmax 300 nm (log ε 3.5); 1H and 13C NMR data, see Tables 1 and 2; HRESIMS: [М-Н]- at m/z: 477.2289. C29H33O6 requires 477.2283.
data, see Tables 1 and 2; HRESIMS: [М-Н]- at m/z: 463.2139. C28H31O6 requires 463.2126. 3.4.3. 6-Methylpatulone (1,2-dihydro-3,8-dihydroxy-6-methoxy1,1-bi (3-methylbut-2-enyl)xanthen-2,9-dione) (3) Yellowish oil; UV (MeOH) lmax 310 nm (log ε 3.4); 1H and 13C NMR data, see Tables 1 and 2; HRESIMS: [М-Н]- at m/z: 409.1662. C24H25O6 requires 409.1657. 3.4.4. (200 R/S)-kellerine C ((R/S)-1,3,5-trihydroxy-2-(3-methyl-2buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3-enyl]-6methoxy-9H-xanthen-9-one) (4) Yellowish oil; ½a25 D þ8 (c 0.1); UV (MeOH) lmax 250 nm (log ε 4.6), 330 nm (log ε 4.3); 1H and 13C NMR data, see Table 3; CDspectrum see supplementary material; HRESIMS: [М-Н]- at m/z: 477.2286. C29H33O6 requires 477.2283. 3.4.5. (200 R/S)-creticine ((R/S)-1,3,6-trihydroxy-5-methoxy-2-(3methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3enyl]-11Н-dibenzo[b,e][1,4]dioxepin-9-one) (5) Brownish-orange oil; ½a25 D -6 (c 0.2, MeOH); UV (MeOH) lmax 1 13 (log ε) 270 (4.0); H and C NMR data, see Table 3; CD-spectrum see supplementary material; HRESIMS: [М-Н]- at m/z: 493.2236. C29H33O7 requires 493.2232. Acknowledgements
3.4.2. Kellerine B (1,2-dihydro-3,6,8-trihydroxy-1,1,5-tri(3methylbut-2-enyl)xanthen-2,9-dione) (2) Yellowish Oil; UV (MeOH) lmax 300 (log ε 3.7); 1H and 13C NMR
We thank F. Kastner, A. Schramm and G. Stühler for measuring the NMR (Bruker Avance III 600 kryo and Bruker Avance III HD 400),
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
€llner for recording the MS spectra (all as well as J. Kiermeier and So Central Analytics of NWF IV, University of Regensburg). PD Dr. A. Dürkop (Institute of Analytical Chemistry of NWF IV, University of Regensburg), J. Ziegler and S. Wiesneth (both Institute of Pharmaceutical Biology of NWF IV, University of Regensburg) are acknowledged for performing the CD experiments. We are indebted to Ms. Gabi Brunner for excellent technical assistance and performing the cytotoxicity assays. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.phytochem.2017.11.009. References Athanasas, K., Magiatis, P., Fokialakis, N., Skaltsounis, A.L., Pratsinis, H., Kletsas, D., 2004. Hyperjovinols A and B: two new phloroglucinol derivatives from Hypericum jovis with antioxidant activity in cell cultures. J. Nat. Prod. 67, 973e977. Avato, P., 2005. A survey of the Hypericum genus: secondary metabolites and bioactivity. Stud. Nat. Prod. Chem. 30, 603e634. Azebaze, A.G.B., Meyer, M., Valentin, A., Nguemfo, E.L., Fomum, Z.T., Nkengfack, A.E., 2006. Prenylated xanthone derivatives with antiplasmodial activity from Allanblackia monticola Staner L.C. Chem. Pharm. Bull. 54, 111e113. Battistini, I., 2013. Analisi LC-PDA-MS di estratti di specie di Hypericum raccolte in Tunisia: studio fitochimico dell',estratto metanolico di Hypericum humifusum L. (Hypericaceae), 2013, Tesi di laurea specialistica LC5, URN etd12182012144131. Chang, S.W., Kim, H.K., Lee, I.K., Sang, U.C., Ryu, S.Y., Kang, R.L., 2009. Phytochemical constituents of Bistorta manshuriensis. Nat. Prod. Sci. 15, 234e240. Crockett, S.L., Robson, N.K.B., 2011. Taxonomy and chemotaxonomy of the genus Hypericum. Med. Arom. Plant. Sci. Biotechnol. 5, 1e13. Crockett, S.L., Wenzig, E.-M., Kunert, O., Bauer, R., 2008. Anti-inflammatory phloroglucinol derivatives from Hypericum empetrifolium. Phytochem. Lett. 1, 37e43. Ernst, E., 2003. The Genus Hypericum L. CRC Press. Farinha, A., Martins, J.M., Nogueira, T., Tavares, R., Duarte, F.A., 2002. HPLC analysis of Hypericum L. Species from Portugal, natural products in the new millennium: prospects and industrial application. In: Volume 47 of the Series Proceedings of the Phytochemical Society of Europe, pp. 125e134. Heilmann, J., Wasescha, M.R., Schmidt, T.J., 2001. The influence of glutathione and cysteine levels on the cytotoxicity of helenanolide type sesquiterpene lactones against KB cells. Bioorg. Med. Chem. 9, 2189e2194. Hyun, S.K., Jung, H.A., Min, B.-S., Jung, J.H., Choi, J.S., 2010. Isolation of phenolics, nucleosides, saccharides and an alkaloid from the root of Aralia cordata. Nat. Prod. Sci. 16, 20e25. Ishiguro, K., Nagareya, N., Suitani, A., Fukumoto, H., 1997. A prenylated xanthone from cell suspension cultures of Hypericum patulum. Phyto¢hemistry 44, 1065e1066. Ito, C., Itoigawa, M., Mishina, Y., Tomiyasu, H., Litaudon, M., Cosson, J.-P., Mukainaka, T., Tokuda, H., Nishino, H., Furukawa, H., 2001. Cancer chemopreventive agents. New depsidones from Garcinia plants. J. Nat. Prod. 64, 147e150. Karioti, A., Vincieri, F.F., Bilia, A.R., 2009. Rapid and efficient purification of naphthodianthrones from St. John's wort extract by using liquideliquid extraction and SEC. J. Sep. Sci. 32, 1374e1382. Karioti, A., Bilia, A.R., 2010. Hypericins as potential leads for new therapeutics. Int. J. Mol. Sci. 11, 562e594.
7
Kitanov, G.M., Nedialkov, P.T., 1998. Mangiferin and isomangiferin in some Hypericum species. Biochem. Syst. Ecol. 26, 647e653. Lebreton, P., Bouchez, M.-P., 1967. Recherches chimiotaxonomiques sur les plantes te rosides polyphe noliques du genre Hypericum. Plant. Med. vasculaires. 9. Les he Phytother. 1, 188e200. Mabry, T.G., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer-Verlag, Berlin, Heidelberg, New York. Makovetska, O.Y., 2000. Research on biologically active substances of Hypericum L. species. Report VII. Sections Drosocarpium Spach., Oligostema (Boiss.) Stef., Thasia Boiss. and Crossophyllum Spach. Farmatsevtichnii Zhurnal (Kiev) 3, 53e59. Neu, R., 1957. Chelate von Diarylbors€ auren mit aliphatischen Oxyalkylaminen als Reagenzien für den Nachweis von Oxyphenyl-benzo-g-pyronen. Die Naturwiss. 44, 181. Ngoupayo, J., Tabopda, T.K., Muhammad, S.A., Tsamo, E., 2008. a-Glucosidase inhibitors from Garcinia brevipedicellata (Clusiaceae). Chem. Pharm. Bull. 56, 1466e1469. Nürk, N.M., Crockett, S.L., 2011. Morphological and phytochemical diversity among Hypericum species of the Mediterranean Basin. Med. Aromatic Plant Sci. Biotechnol. 5 (Special Issue 1), 14e28. Permanaa, D., Abas, F., Maulidiani, Shaari K., Stanslas, J., Ali, A.M., Lajis, N.H., 2005. Atrovirisidone B, a new prenylated depsidone with cytotoxic property from the roots of Garcinia atroviridis. Z. Naturforsch. 60c, 523e526. Rios, M.Y., Delgado, G., 1992. Polyprenols and acylphloroglucinols from Esenbeckia nesiotica. Phytochemistry 31, 3491e3494. Robson, N.K.B., 2010. Studies in the genus Hypericum L. (Hypericaceae) 5(1). Sections 10. Olympia to 15/16. Crossophyllum. Phytotaxa 4, 5e126. Rouis, Z., Abid, N., Aouni, M., Faiella, L., Dal Piaz, F., De Tommasi, N., Braca, A., 2013. Benzophenone glycosides from Hypericum humifusum ssp. australe. J. Nat. Prod. 76, 979e982. Schmidt, S., Jürgenliemk, G., Skaltsa, H., Heilmann, J., 2012a. Phloroglucinol derivatives from Hypericum empetrifolium with antiproliferative activity on endothelial cells. Phytochemistry 77, 218e225. Schmidt, S., Jürgenliemk, G., Schmidt, T.J., Skaltsa, H., Heilmann, J., 2012b. Bi-, tri-, and polycyclic acylphloroglucinols from Hypericum empetrifolium. J. Nat. Prod. 75, 1697e1705. Seabra, R.M., Alves, A.C., 1989. Mangiferin and chlorogenic acid from Hypericum species. Planta Medica 55, 404. Tanaka, N., Mamemura, T., Abe, S., Imabayashi, K., Kashiwada, Y., Takaishi, Y., Suzuki, T., Takebe, Y., Kubota, T., Kobayashi, J., 2010. Biyouxanthones A - D, prenylated xanthones from roots of Hypericum chinense. Heterocycles 80, 613e621. Tatsis, E.C., Boeren, S., Exarchou, V., Troganis, A.N., Vervoort, J., Gerothanassis, I.P., 2007. Identification of the major constituents of Hypericum perforatum by LC/ SPE/NMR and LC/MS. Phytochemistry 68, 383e393. Tatsis, E.C., Exarchou, V., Troganis, A.N., Gerothanassis, I.P., 2009. 1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups. Anal. Chim. Acta 607, 219e226. Trigas, P., Panitsa, M., Tsiftsis, S., 2013. Elevational gradient of vascular plant species richness and endemism in Crete e the effect of post-isolation mountain uplift on a continental island system. PLoS ONE 8, e59425. Umek, A., Kreft, S., Kartnig, T., Heydel, B., 1999. Quantitative phytochemical analyses of six Hypericum species growing in Slovenia. Planta Med. 65, 388e390. Winkelmann, K., San, M., Kypriotakis, Z., Skaltsa, H., Bosilij, B., Heilmann, J., 2003. Antibacterial and cytotoxic activity of prenylated bicyclic acylphloroglucinol derivatives from Hypericum amblycalyx. Z. Naturforsch. 58c, 527e532. Xu, Y.-J., Chiang, P.-Y., Lai, Y.-H., Vittal, J.J., Wu, X.-H., Tan, B.K.H., Imiyabir, Z., Goh, S.H., 2000. Cytotoxic prenylated depsidones from Garcinia parvifolia. J. Nat. Prod. 63, 1361e1363. Xu, W.-J., Li, R.-J., Quasie, O., Yang, M.-H., Kong, L.-Y., Luo, J., 2016. Polyprenylated tetraoxygenated xanthones from the roots of Hypericum monogynum and their neuroprotective activities. J. Nat. Prod. 79, 1971e1981.
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Phenolic metabolites from Hypericum kelleri BALD., an endemic species of Crete (Greece) Angeliki Mathioudaki a, b, Ariola Berzesta a, b, Zacharias Kypriotakis c, Helen Skaltsa a, €rg Heilmann b, * Jo a
Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis, Zografou, 157 71, Athens, Greece €t Regensburg, Pharmaceutical Biology, Universita €tsstr. 31, D-93053, Regensburg, Germany Universita c Technological Education Institute, School of Agricultural Production, Lab. of Taxonomy and Management of Wild Flora, Stavromenos P.O.Box 140, Heraklion-Crete, 71110, Greece b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2017 Received in revised form 13 November 2017 Accepted 22 November 2017
Thirteen compounds were isolated from the aerial parts of Hypericum kelleri BALD., growing as an endemic on the island of Crete (Greece). These compounds comprise four previously unknown prenylated xanthones 1,2-dihydro-3,8-dihydroxy-6-methoxy-1,1,5-tri(3-methylbut-2-enyl)xanthen-2,9-dione (kellerine A), 1,2-dihydro-3,6,8-trihydroxy-1,1,5-tri(3-methylbut-2-enyl)xanthen-2,9-dione (kellerine B), 1,2dihydro-3,8-dihydroxy-6-methoxy-1,1-bi(3-methylbut-2-enyl)xanthen-2,9-dione (6-methylpatulone), (R/S)-1,3,5-trihydroxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3-enyl]-6methoxy-9H-xanthen-9-one ((200 R/S)-kellerine C) and the hitherto undescribed depsidone (R/S)-1,3,6trihydroxy-5-methoxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3-enyl]-11Нdibenzo[b,e] [1,4]dioxepin-9-one ((200 R/S)-creticine). As known compounds, brevipsidone D, 4-geranyl-2(20 -isobutyryl)-phloroglucinol, 4-geranyl-2-(20 -methylbutyryl)-phloroglucinol, I3, II8-biapigenin, quercetin, avicularin, pseudohypericin and neochlorogenic acid have been isolated. The structures were elucidated on the basis of their 1D, 2D NMR, CD and MS data. The study confirms the typical occurrence of xanthones in Hypericum section Oligostema (BOISS.) STEF., and is also the first report on the simultaneous isolation of acylphloroglucinols in this section. Furthermore the first evidence of depsidones in the genus Hypericum L. is reported. Cytotoxicity was investigated in HeLa cells for prenylated xanthones and the depsidones. Both triprenylated 1,2-dihydroxanthones (kellerine A and B) showed significant in vitro cytotoxicity with IC50 values of 2.5 ± 0.1 (kellerine A) and 5.9 ± 0.9 (kellerine B) mM, whereas other compounds were less cytotoxic (IC50 > 20 mM). © 2017 Published by Elsevier Ltd.
Dedicated to Prof. Dr. Otto Sticher on the occasion of his 80th birthday and as homage to his outstanding phytochemical and analytical work. Keywords: Hypericum kelleri Hypericaceae Xanthones Kellerines A-C Chemotaxonomy Depsidones Creticine
1. Introduction Hypericum L. is a large genus with pharmaceutically relevant representatives, which comprises ~470 species of trees, shrubs, and herbs οf worldwide distribution, but most species originate from temperate regions and tropical mountains (Crockett and Robson, 2011). Several of them have been the subject of chemical and pharmacological investigations and proved a rich source of acylphloroglucinols, xanthones and flavonoids (Ernst, 2003; Avato, 2005; Karioti and Bilia, 2010).
* Corresponding author. E-mail address: [email protected] (J. Heilmann). https://doi.org/10.1016/j.phytochem.2017.11.009 0031-9422/© 2017 Published by Elsevier Ltd.
The present study deals with the first phytochemical investigation of H. kelleri BALD., (Hypericaceae) belonging to the section Oligostema (BOISS.) STEF., a narrow endemic species in western Crete. The flora of the island includes 1825 native vascular plant species, 10.6% of which are single island endemic species, with a distribution range restricted to the main island of Crete (Trigas et al., 2013). The Cretan taxons of Hypericum share a similar habitus as dwarf shrubs and showed a preferred distribution in the low mountain range. Nevertheless, they exhibit different morphology with regards to glands and leaves and belong to different sections. Three species of the section Coridium namely H. empetrifolium (Schmidt et al., 2012a and b), H. amblycalyx COUST. & GAND. (Winkelmann et al., 2003) and H. jovis GREUT. (Athanasas et al., 2004), have been phytochemically investigated, all showing
2
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
prenylated mono, di- and tricyclic acylphloroglucinols as typical specialized metabolites. Most of these generates from an activated isobutyric or 2-methylbutyric acid as biosynthetic starter molecule. In contrast, H. aciferum (GREUT) N. ROBSON (sect. Adenotrias), H. trichocaulon BOISS. & HELDR. (sect. Drosocarpium) and H. kelleri (sect. Oligostema) have not been investigated up to now.
2. Results and discussion The cyclohexane extract of the aerial parts of Hypericum kelleri BALD. was fractionated by centrifugal partition chromatography followed by silica column chromatography and semi-preparative HPLC and yielded previously undescribed xanthones (Fig. 1) 1,2dihydro-3,8-dihydroxy-6-methoxy-1,1,5-tri(3-methylbut-2-enyl) xanthen-2,9-dione (kellerine A, 1), 1,2-dihydro-3,6,8-trihydroxy1,1,5-tri(3-methylbut-2-enyl)xanthen-2,9-dione (kellerine B, 2), 1,2-dihydro-3,8-dihydroxy-6-methoxy-1,1-bi(3-methylbut-2-enyl) xanthen-2,9-dione (6-methyl-patulone, 3), (R/S)-1,3,5-trihydroxy2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-
methylbut-3-enyl]-6-methoxy-9H-xanthen-9-one ((200 R/S)-kellerine C, 4), as well as the unknown depsidone (R/S)-1,3,6-trihydroxy5-methoxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2enyl)-3-methylbut-3-enyl]-11Н-dibenzo[b,e] [1,4]dioxepin-9-one ((200 R/S)-creticine, 5) and known brevipsidone D (6) (Ngoupayo et al., 2008). The dichloromethane extract of the same plant material was fractionated by flash chromatography, CPC (Centrifugal partition chromatography) and RP18-HPLC and yielded 4-geranyl2-(20 -isobutyryl)-phloroglucinol (7) (Crockett et al., 2008; Rios and Delgado, 1992), 4-geranyl-2-(20 -methylbutyryl)-phloroglucinol (8) (Crockett et al., 2008; Rios and Delgado, 1992). Finally, the methanol extract was fractionated by liquid-liquid extraction and column chromatography on Sephadex® LH-20 and afforded quercetin (9) (Tatsis et al., 2007; Chang et al., 2009), avicularin (10) (Chang et al., 2009), I3,II8-diapigenin (11) (Tatsis et al., 2007), pseudohypericin (12) (Tatsis et al., 2009; Karioti et al., 2009) and neochlorogenic acid (13) (Hyun et al., 2010). The structures of the compounds were established on the basis of 1D and 2D NMR as well as CD and HRMS techniques. Compounds
Fig. 1. Structures of compounds 1e5.
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
1e4 were isolated as yellow oily residues and showed similar NMR features with scaffold characteristic signals referring to xanthone derivatives. Compound 1 had a molecular formula of C29H34O6 based on HRESIMS m/z 477.2289 [M-H]-. The 1H NMR spectrum of 1 showed the presence of a hydrogen-bonded hydroxyl proton at dH 13.23 (1H, s, 8-ΟН), two aromatic protons at dH 6.51 (1H, s, Н-4) and 6.39 (1H, s, Н-7), one methoxy-group at dH 3.89 (3H, s, 6-OCH3) and one hydroxyl proton at dH 7.00 (1H, s, 3-OH). In addition, the 1H NMR spectrum revealed signals at dН 4.64 (2Н, t, J ¼ 7.6 Hz, H-20 and H-200 ), 3.43 (2H, dd, J ¼ 7.6, 13.7 Hz, H-1a0 and H-1a00 ), 2.80 (2H, dd, J ¼ 7.6, 13.7 Hz, H-1b0 and H-1b00 ), 1.48 (6Н, s, H-40 and H-400 ) and 1.49 (6H, s, H-50 and H-500 ) characteristic of the presence of gem di (3-methylbut-2-enyl)-side chains (Azebaze et al., 2006). Furthermore, the rest of the signals at dН 3.43 (2H, d, J ¼ 7.2 Hz, H-1000 ), 5.21 (1H, t, J ¼ 7.2 Hz, H-2000 ), 1.83 (3Н, s, Н-4000 ) and 1.69 (3H, s, Н-5000 ) revealed the presence of one more 3-methylbut-2-enyl lateral chain connected. These data suggested that 1 is a xanthone derivative having three prenyl side chains. 1H,1H COSY spectrum confirms the proton correlations within the prenyl sidechains and 1 13 H, C HSQC enable the respective assignment of the methines, methylenes and methyl groups. In the 1H,13C HMBC spectrum, the following key long-range correlations were observed: OH-3 only with C-4 (dC 108.9) while H-4 correlated with C-2 (dC 201.3), C-3 (dC 151.7), C-4a (dC 159.2), C-9 (dC 179.8), C-9a (dC 116.2); 8-ΟН with C-6 (dC 162.6), C-7 (dC 95.3), C- 8 (dC 161.1), C-8a (dC 104.7); 6-OCH3 with C-6. The long-range correlations of the CH2-10 and 100 protons with C-1 (dC 55.9) and C-2 revealed that the gem 3-methylbut-2-enyl lateral chains are located at C-1. Furthermore, the HMBC cross peaks between CH2-1000 and C-4b (dC 153.3), C-5 (dC 107.2) and C-6 revealed that the third 3-methylbut-2-enyl side chain is attached to C-5 (dC 107.2). Methoxylation at C-6 was verified by 1H,1H ROESY correlations of methyl protons with H-7 and the observed hydrogen bond of hydroxyl group at C-8. On the basis of these data the hitherto unknown structure of 1 was determined as 1,2-dihydro3,8-dihydroxy-6-methoxy-1,1,5-tri(3-methylbutyl-2-enyl) xanthen-2,9-dione and named kellerine A. The 1H and 13C NMR spectroscopic data of compound 2, C28H32O6, were very similar to those of 1 except for the substituent at C-6. The 1H NMR data showed the absence of the signal at dН 3.89 (3H, s) and the presence of one hydroxyl group at dН 5.95 (1H, s, 6OH); HMBC cross peaks with C-5 (dC 104.7), C-6 (dC 160.7) and C-7 (dC 99.6) confirmed the site of attachment. 1H,1H COSY and 1H,13C HSQC spectra also matched the 2D NMR analysis of 1. Thus, compound 2 was also isolated for the first time and determined as 1,2dihydro-3,6,8-trihydroxy-1,1,5-tri(3-methylbut-2-enyl)xanthen2,9-dione and was named kellerine B. The main difference at the 1H and 13C NMR spectroscopic data of compound 3, C24H26O6, compared to 1, is the absence of the signals corresponding at the 3-methylbutyl-2-enyl side chain of C-5. Instead, in the 1H NMR spectrum, one more methine at dН 6.39 (H5) appeared as doublet with meta coupling with H-7 (dН 6.36, J ¼ 2.2), while in 13C NMR, C-5 (dC 92.2) resonated upfield compared with 1 and 2. This was confirmed by an additional crosspeak (dН 6.36/6.39) in 1H,1H COSY and an additional crosspeak for dH/C 6.39/ 92.2 in 1H,13C HSQC spectrum. Thus, previously undescribed compound 3 was determined as 1,2-dihydro-3,8-dihydroxy-6methoxy-1,1-bi (3-methylbut-2-enyl)xanthen-2,9-dione and was named 6-methyl-patulone based on the structural similarities with the previously isolated patulone (Ishiguro et al., 1997). Compound 4, obtained as a yellow oily residue, showed a molecular formula of C29H34O6 on the basis of HRESIMS m/z 477.2286 [M-H]-. The 1H NMR spectrum showed the characteristic signals of two ortho-aromatic protons at dН 6.97 (1Н, d, J ¼ 8.8 Hz, H-7) and 7.81 (1H, d, J ¼ 8.8 Hz, H-8), one hydrogen-bonded hydroxyl proton at dH 13.31 (1H, s), two phenolic hydroxyl groups at dH 6.28 (1H, s,
3
3-OH) and 5.63 (1H, s, 5-OH), one methoxy group at dH 4.04 (3H, s), one prenyl group [dH 3.48 (2H, d, J ¼ 7.2 Hz, H-10 ); dH 5.27 (1H, t, J ¼ 7.2 Hz, H-20 ); dH 1.86 (3H, s, H-40 ) and dH 1.78 (3H, s, H-50 )], and a 2-(3-methylbut-2-enyl)-3-methylbut-3-enyl-group [dH 2.91 (2H, d, J ¼ 7.2 Hz, H-100 ); dH 2.51 (1H, quint, J ¼ 7.2 Hz, H-200 ); dH 2.19/2.18 (2H, t, J ¼ 7.1 Hz, H-1000 ); dH 5.13 (1H, t, J ¼ 7.1 Hz, H-2000 ); dH 1.58 (3H, s, H-4000 ); dH 1.68 (3H, s, H-5000 ); dH 4.64/4.68 (2H, brs, H-400 ) and dH 1.75 (3H, s, H-500 )]. The 13C spectra displayed signals of 29 carbons including 14 quaternary, 5 methine, 4 methylene, and 6 methyl carbons, which could be well assigned to a substituted xanthone skeleton. The seven substituents on the xanthone skeleton were determined on the basis of 1H,13C HMBC, 1H,1H COSY and 1H,13C HSQC spectral analysis. In the HMBC spectrum, the hydrogenbonded hydroxyl proton correlated with C-1 (dC 158.4); C-2 (dC 108.4) and C-9a (dC 102.7). The prenyl group was located at C-2, since its typical methylene protons at dH 3.48 showed HMBC correlations with C-1 (dC 158.4); C-2 (dC 108.4); C-3 (dC 160.7); C-20 (dC 121.3) and C-30 (dC 136.0). The presence of a 2-(3-methylbut-2enyl)-3-methylbut-3-enyl-group was elucidated by 1H,1H COSY and 1H,13C HSQC correlations. It is located at C-4, since the methylene protons at dH 2.91 showed HMBC correlations with C-4 (dC 106.1); C-4a (dC 149.0) and C-3 (dC 160.7) and the methylene protons at dH 2.18/2.19 showed HMBC correlations with C-4a (dC 149.0, J5). The protons of the methoxy group correlated with C-6 (dC 151.1). The ortho-aromatic protons at dH 6.97 and 7.81 were assigned as H7 and H-8, respectively, according to the coupling between the proton at dH 7.81 and the carbonyl carbon C-9 (dC 180.9). The two phenolic hydroxyl groups at dH 6.28 (1H, s) and 5.63 (1H, s) correlated with C-3 (dC 160.7) and C-5 (dC 133.4), respectively. To determine the absolute stereochemistry for C-200 , a CD-spectrum was recorded. In comparison to experimental CD data gained for the very similar xanthones monogxanthone A and B (Xu et al., 2016) with the concerning C being R-configurated, kellerine C must be racemic due to lacking Cotton effects. Thus, the structure of 4 was identified as the previously unknown (R/S)-1,3,5-trihydroxy2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3methylbut-3-enyl]-6-methoxy-9H-xanthen-9-one and was named (200 R/S)-kellerine C. The 6-desmethylderivative of kellerine C was previously isolated from H. monogynum L. (syn. H. chinense, not accepted) by Tanaka et al. (2010) and Xu et al. (2016) and named biyouxanthone D. Compound 5, obtained as a yellow oily residue, showed a molecular formula of C29H34O7 based on HRESIMS m/z 493.2236 [MH]-. The proton signals in the 1H NMR spectrum were quite similar to the signals of compound 4 showing again the characteristic signals of two ortho-aromatic protons at dН 6.75 (1Н, d, J ¼ 9.1 Hz, H-7) and 6.93 (1H, d, J ¼ 9.1 Hz, H-8), two phenolic hydroxyl groups at dH 6.27 (1H, s, 3-OH) and 5.53 (1H, s, 6-OH), one methoxy group at dH 3.99 (3H, s), one prenyl group [dH 3.40 (2H, d, J ¼ 7.1 Hz, H-10 ); dH 5.17 (1H, t, J ¼ 7.1 Hz, H-20 ); dH 1.81 (3H, s, H-40 ) and dH 1.66 (3H, s, H-50 )], and a 2-(3-methylbut-2-enyl)-3-methylbut-3-enyl-group [dH 3.00 (2H, d, J ¼ 7.4 Hz, H-100 ); dH 2.51 (1H, quint, J ¼ 7.4 Hz, H200 ); dH 2.14 (2H, t, J ¼ 6.9 Hz, H-1000 ); dH 5.04 (1H, t, J ¼ 6.9 Hz, H-2000 ); dH 1.56 (3H,s, H-4000 ); dH 1.66 (3H, s, H-5000 ); dH 4.78/4.71 (2H, brs, H400 ) and dH 1.75 (3H, s, H-500 )]. 1H,1H COSY and 1H,13C HSQC spectra also showed matching correlations. The only main difference was regarding the 1H shift of the hydrogen-bonded hydroxyl proton, which resonated upfield shifted at dH 11.08 ppm together with the downfield shift of the aromatic protons H-7 and H-8. In addition, dC values at the 13C spectra of compounds 4 and 5 were quite similar, except of the C-8a, which was de-shielded to dC 138.7 ppm. The molecular formula of compound 5 confirms that an additional oxygen causes this de-shielding. Accordingly, C-9 of 5 is not an unsaturated keto functionality, but now an ester showing an upfield shift from dC 180.9 in 4 to dC 168.0 ppm in 5. Again, lacking Cotton
4
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
effects in ECD spectrum and comparison to experimental and calculated CD data (Xu et al., 2016) showed the presence of an racemate. Thus, compound 5 was identified as the previously undescribed depsidone derivative (R/S)-(1,3,6-trihydroxy-5methoxy-2-(3-methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)3-methylbut-3-enyl]-11Н-dibenzo[b,e][1,4]dioxepin-9-one and was named (200 R/S)-creticine. H. kelleri BALD. belongs to the section Oligostema (BOISS.) Stef., together with H. andjerinum Font Quer & Pau, H. australe TEN (¼H. humifusum subsp. australe (TEN.) ROUY & FOUCAUD ¼ H. linarifolium BERTOL.), H. humifusum L., H. linearifolium WILLD. (¼ H. linarifolium VAHL ¼ H. humifusum subsp. linarifolium (Vahl) ROUY & FOUCAUD) and H. repens L. (¼ H. tenellum E. D. CLARKE) based on similarities in floral and vegetative morphology (Robson, 2010; Nürk and Crockett, 2011). Few phytochemical studies have been carried out for most of these species revealing the presence of xanthones (i.e. mangiferin/isomangiferin), flavonoids, phenolic acids and naphthodianthrones. This is the first phytochemical study on H. kelleri. Concerning the presence of xanthones, our results are in agreement to previous studies in Oligostema; despite the fact that they occur as aglycones and not as C-glycosides in H. kelleri, it seems to be a typical class of specialized metabolites in this section. It is noteworthy that kellerines are oxidized (and methoxylated) derivatives of tetrahydroxy xanthones and therefore structurally closely related to mangiferin, so far, found in H. humifusum (Kitanov and Nedialkov, 1998; Battistini, 2013), H. humifusum subsp. australe (Rouis et al., 2013) and isomangiferin in H. linearifolium (Seabra and Alves, 1989). Moreover, 4-geranyl-2(20 -isobutyryl)-phloroglucinol and 4-geranyl-2-(20 -methylbutyryl)-phloroglucinol found in H. kelleri are the first isolated acylphloroglucinols from Oligostema, to the best of our knowledge. Furthermore, this study is the first evidence of depsidones in the genus Hypericum L. and just the second one into the Plantae kingdom, while they have been isolated only from the genus Garcinia till today (Permanaa et al., 2005; Ito et al., 2001; Xu et al., 2000). Among the different groups of specialized metabolites present in Hypericum L. the polyphenolic metabolites pseudohypericin, hypericin as naphtodianthrons, quercetin, isoquercitrin, rutin as flavonoids and the phenylpropanoids chlorogenic and neochlorogenic acids have been also often investigated. Previous studies in taxa belonging to Oligostema revealed their presence as follows: hypericin has been found in H. humifusum (Umek et al., 1999; Farinha et al., 2002), H. linearifolium (Farinha et al., 2002) and H. repens (Makovetska, 2000); pseudohypericin in the latter two species (Makovetska, 2000; Farinha et al., 2002); quercetin and hyperoside in H. humifusum (Lebreton and Bouchez, 1967; Umek et al., 1999; Farinha et al., 2002), as well as quercitrin, isoquercetrin, amentoflavone, I3,II8-biapigenin in H. humifusum (Umek et al., 1999; Farinha et al., 2002), while in H. linearifolium have been found all the previous quoted flavonoids, except the last one, plus rutin (Farinha et al., 2002). Moreover, chlorogenic acid has been found in H. humifusum, H. linearifolium (Rouis et al., 2013); its methyl ester, neochlorogenic acid and 3-O-p-coumaroyl quinic acid in H. humifusum ssp. australe (Rouis et al., 2013; Battistini, 2013). Although these compounds are not considered as chemotaxonomic markers, they are related to the pharmacological effects of the plants (Crockett and Robson, 2011). Consequently, we also investigated the methanol fractions and were able to isolate the naphthodianthrone pseudohypericin (12), the flavonoids quercetin (9), avicularin (10) and I3,II8-biapigenin (11), as well as neochlorogenic acid (13). Extensive TLC analyses of enriched Sephadex-LH20 fractions confirm the lack of traceable amounts of hypericin, kaempferol, isoquercitrin, hyperoside, rutin and amentoflavone. It is worth mentioning that during the whole isolation course, main
constituents, as well as minor ones were continuously monitored and traced down using an NMR metabolomic strategy, which permitted in detail characterization of extracts and sub fractions thereof. Cytotoxicity was evaluated for hitherto unknown compounds (1e5) and brevipsidone D in HeLa cells. Only the 1,2dihydroxanthones substituted by three prenyl groups (1 and 2) showed significant in vitro cytotoxicity with IC50 values of 2.5 ± 0.1 (1) and 5.9 ± 0.9 (2) mM, whereas other compounds were less cytotoxic (IC50 > 20 mM). 3. Experimental section 3.1. General experimental procedures Optical rotations were recorded using a UniPol L1000 polarimeter (Schmidt þ Haensch) and a Perkin-Elmer 341 Polarimeter. UV spectra were obtained in MeOH on a Cary 50 Scan spectrophotometer (Varian) according to Mabry et al. (1970). CD-data were gained in MeOH on a J-715 spectropolarimeter (JASCO) at 22 C from 200 to 400 nm with the compounds concentrated with 200 mmol/L. 1H, 13C and 2D NMR spectra were recorded in CDCl3 and CD3OD on Bruker Avance III 600 equipped with a 5 mm TCI CryoProbe, Bruker DRX 400, Bruker Avance III HD 400 and Bruker AC 200 instruments at 295 K. Chemical shifts are given in ppm (d) and were referenced to the solvent signals at 7.24/3.31 and 77.0/ 49.0 ppm for 1H and 13C NMR, respectively. 1H,1H COSY, 1H,13C HSQC, 1H,13C HMBC, and 1H,1H ROESY (mixing time 950 ms) were performed using standard Bruker microprograms. High resolution mass spectra were measured on a Finnigan MAT SSQ 710 A at 70 eV (HREIMS, positive mode) or recorded on an Agilent 6540 UHD (HRESIMS, positive and negative mode). The solvents used were of spectroscopical grade (Merck). Column chromatography: silica gel (Geduran® Si 60 for column chromatography; Merck), gradient elution with the solvent mixtures indicated in each case. Semipreparative HPLC separations were performed on a Varian ProStar 210 Solvent Delivery Module equipped with a Varian ProStar 335 Photodiode Array Detector using as RP-18 HPLC column an Eclipse XDB-C18 (5 mm, 9.4 250 mm, Agilent, USA). All solvents used were of HPLC grade (Merck). Centrifugal partition chromatography was performed on an Armen Instrument Spot CPC (Alpha Crom, Laboratory equipment), with a 515 HPLC-pump (Waters, USA) and the fractions were collected automatically by a LKM Bromma 2211 Superrac instrument (SE). All solvents used were of p.a. grade (Merck). Silica gel 60 F254 precoated aluminium sheets and silica gel 60 RP-18 F254s precoated sheets (both from Merck) were used for TLC. Fractionation was always monitored by TLC silica gel 60 F-254, (Merck, Art. 5554) and silica gel 60 RP-18 F254s (Merck, Art. 15685) plates with visualization under UV (254 and 365 nm) and spraying with anisaldehyde-sulfuric acid reagent (anisaldehyde, 2 mL; concentrated H2SO4, 10 mL; HOAc, 16 mL; MeOH, 170 mL), macrogol 400 reagent (5 g PEG; 100 mL MeOH) and with Neu’ s reagent for phenolics (Neu, 1957). 3.2. Plant material Aerial parts of H. kelleri BALD. ((Hypericaceae) were collected on the whole plateau of Omalos (35 200 3700 N, 23 540 100 ; ~1040e1250 m), in the western part of Crete in June 2013 (regional district Chania). The plant was identified by Dr. Z. Kypriotakis. A voucher specimen is deposited at the Herbarium of Technological Education Institute, School of Agricultural Production, Lab. of Taxonomy and Management of Wild Flora (Heraklion-Crete, Greece) with the identification number 15925/26-6-2013.
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
5
Table 1 1 Н NMR of compounds 1e3 (CDCl3, 600 MHz, 295 K).Chemical shifts are given in ppm and J in Hz. Position
1
HMBC
2
HMBC
3
HMBC
4 5 7 1a0 , 1a00 1b0 , 1b00 20 , 200 40 , 400 50 , 500 1000 2000 4000 5000 3-OH 6-ΟR 8-ΟН
6.51 s e 6.39 s 3.43 dd (7.6, 13.7) 2.80 dd (7.6, 13.7) 4.64 t (7.6) 1.48 s 1.49 s 3.43 d (7.2) 5.21 t (7.2) 1.83 s 1.69 s 7.00 s 3.89 s 13.23 s
2, 3, 4a, 9, 9a e 5, 6, 8a, 8 1, 2, 9a, 10 , 20 , 30 ,100 , 200 , 300
6.51 s e 6.30 s 3.44 dd (7.6, 13.7) 2.80 dd (7.6, 13.7) 4.64 t (7.6) 1.48 s 1.49s 3.52 d (7.2) 5.30 t (7.2) 1.87 s 1.78 s 6.95 s 5.95 s 13.14 s
2, 3, 4a, 9, 9a e 8a, 8 1, 2, 10 , 20 , 30 , 100 , 200 , 300
6.49 s 6.39 d (2.2) 6.36 d (2.2) 3.44 dd (7.7, 13.8) 2.79 dd (7.7, 13.8) 4.63 t (7.6) 1.48 s 1.48 s e e e e 6.96 s 3.87 s 13.15 s
2, 3, 4a, 9, 9a 4b, 6, 7, 8, 8a 5, 6, 8, 8a 1, 2, 10 , 20 , 30 , 100 , 200 , 300
10 , 40 , 50 , 100 , 400 , 500 1, 10 , 20 , 30 , 50 , 100 , 200 , 300 , 500 1, 10 , 20 , 30 ,40 , 100 , 200 , 300 ,400 4b, 5, 6, 2000 , 3000 1000 , 4000 , 5000 2000 , 3000 , 5000 2000 , 3000 , 4000 4 6 6, 7, 8, 8a
10 , 40 , 50 , 100 , 400 , 500 20 , 30 , 50 ,200 , 300 , 500 20 , 30 ,40 , 200 , 300 ,400 4b, 5, 6, 2000 , 3000 1000 , 4000 , 5000 2000 , 3000 , 5000 2000 , 3000 , 4000 2, 3, 4, 4a 5, 6, 7 7, 8, 8a
10 , 40 , 50 , 100 , 400 , 500 1, 20 , 30 , 50 , 200 , 300 , 500 1, 20 , 30 , 40 , 200 , 300 ,400 e e e e 2, 3, 4 6 7, 8, 8a
1, 3: R¼CH3, 2: R¼H.
3.3. Cell culture and determination of cytotoxicity HeLa cells (ATCC CCL17) were cultured at 37 C in a humidified incubator with 5% CO2. Culture medium was MEM (Biochrom AG) supplemented with 10% FCS and 2 mM L-glutamine. Cytotoxicity was evaluated in an MTT assay assessing the metabolic activity of NADPH-dependent cellular oxidoreductase enzymes capable of reducing MTT to its insoluble formazan as described by Heilmann et al. (2001). Tests were performed in duplicates and all experiments repeated three times (n ¼ 8). IC50 values were calculated from eight different concentrations and data are reported as mean ± SD. Maximal observed (absolute) standard deviation was about 15%. Positive control measurements were performed with xanthohumol. 3.4. Extraction and isolation Air-dried and powdered aerial parts of Hypericum kelleri BALD. (258.1 g) were extracted by maceration with cyclohexane, DCM, MeOH, and 5:1 (v/v) MeOH-H2O mixture, successively. The cyclohexane-soluble material (2.64 g) was defatted using MeOH at 0 C and the supernatant layer resulted after removal of solvent under vacuum a residue (2.17 g), which was subjected to centrifugal partition chromatography using a solvent system of MeOH-heptane (1:1) (flow rate 10 mL/min; rotation: 800 rpm; ASC) and resulted 434 fractions. Based on TLC similarities, identical fractions were combined to give a total of 11 fractions (KEC.1KEC.11). Part of KEC.5 (0.010 g) subjected to further purification by RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) using a H2OACN gradient (60% ACN 0e5 min; 60% / 97% ACN 5e20 min; 97% ACN 20e22 min; 97% / 80% ACN 22e23 min; 80% ACN 23e33 min; 80% / 60% ACN 33e34 min; 60% ACN 34e44 min) yielded compound 1 (5.1 mg, tR 28.19). Applying a H2O-ACN gradient (60% ACN 0e5 min; 60% / 97% ACN 5e25 min; 97% ACN 25e30 min; 97% / 60% ACN 30e31 min; 60% ACN 31e41 min) to fraction KEC.6 (8.0 mg) subjected to RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) yielded compound 3 (1.5 mg, tR 21.71). Fraction KEC.9 (17.5 mg) was subjected to RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) using the same gradient system as before to fraction KEC.6 yielded compound 2 (9.1 mg, tR 20.85). Part of fraction KEC.10 (88.1 mg) was applied to column chromatography over silica gel (50.5 1.5 cm) eluted with mixtures of increasing polarity of cyclohexane-EtOAc (flow rate 1.5 mL/min) and yielded 432 fractions (z1.5 mL each), which were combined based on TLC similarities to 11 fractions (KEC.10.1-KEC.10.11) one of which constitutes compound 5 (2.8 mg; fraction KEC.10.2). KEC.10.4 (11.6 mg)
subjected to further purification by RP18-HPLC (Eurosphere-100 C18; flow rate 3 mL/min) using a H2O-ACN gradient (60% ACN 0e5 min; 60% / 97% ACN 5e25 min; 97% ACN 25e30 min; 97% / 60% ACN 30e31 min; 60% ACN 31e41 min) and yielded compound 6 (1.1 mg; tR 21.5) and compound 4 (0.9 mg; tR 26.91). The dichloromethane extract (5.75 g) was defatted by CC through Diaion LH-20 and eluted with MeOH 90%, MeOH 100% and DCM to yield three fractions (DC 1e3). DC-1 was further fractionated by flash chromatography (Geduran® 60) eluted with mixtures of increasing polarity of hexane-EtOAc (flow rate 50 mL/min) and yielded 373 fractions (z23 mL each), which were combined based on TLC similarities to 12 fractions (F1-F12). Fraction F10 was subjected to CPC using hexane-EtOAc-MeOH-H2O 7:3:7:3 (flow rate 5 mL/min; rotation: 1200 rpm) and afforded 312 fractions combined, based on TLC similarities, to 7 sub-groups (F10.1 and F10.7). Fraction F10.2 was subjected on flash chromatography (Lichoprep® RP-18; 26 mm 12 cm) using a H2O-MeOH gradient (50% MeOH 0e2 min; 50% / 100% MeOH 2e60 min; 100% MeOH 60e105 min) and yielded 7 sub-fractions (F10.2.1- F10.2.7). Fraction F10.2.3 was
Table 2 13 C NMR of compounds 1e3 (CDCl3, 150 MHzat 295 K). Chemical shifts are given in ppm. Position
1
2
3
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 /100 20 /200 30 /300 40 /400 50 /500 1000 2000 3000 4000 5000 6-ΟR
55.9 201.3 151.7 108.9 159.2 153.3 107.2 162.6 95.3 161.1 104.7 179.8 116.2 37.9 117.8 135.2 17.9 25.7 21.6 121.9 131.9 17.9 25.8 56.0
55.9 201.4 151.8 108.7 154.0 153.8 104.7 160.7 99.6 160.5 105.1 179.7 116.4 37.9 117.7 135.3 17.9 25.7 21.7 121.2 134.8 18.0 25.8 e
56.1 201.2 151.7 108.7 158.8 156.0 92.2 165.4 98.2 162.6 105.1 179.2 116.9 38.2 117.6 135.5 18.0 25.6
1, 3: R¼CH3, 2: R¼H.
e e e e 55.9
6
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
Table 3 1 Н and 13C NMR of compounds 4 and 5 (CDCl3, 600 MHz for 1H, 150 MHz for a
C
mult
4
5
4
dC
dC
dH
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 20 30 40 50 100 200
C C C C C C C C CH CH C C C CH2 CH C CH3 CH3 CH2 CH
158.4 108.4 160.7 106.1 149.0 144.4 133.4 151.1 107.3 117.0 115.6 180.9 102.7 21.6 121.3 136.0 18.0 25.9 27.6 47.3
160.1 111.0 160.9 112.3 158.6 143.8 138.5 147.6 111.5 116.4 138.7 168.0 98.5 22.3 120.8 136.3 17.9 25.7 27.8 46.9
300 400 500 1000 2000 3000 4000 5000 1-OH 3-OH 5-OH 6-ΟН 5-OCH3 6-OCH3
C CH2 CH3 CH2 CH C CH3 CH3 e e e e CH3 CH3
153.4 111.0 19.3 31.7 123.1 132.5 17.9 25.8 e e e e
148.3 111.4 19.7 30.8 122.7 132.5 18.0 25.8 e e e e 62.9
a
56.6
13
C at 295 K). HMBC of 4
5
HMBC of 5
dH
6.97 d (8.8) 7.81 d (8.8)
4b, 5, 6, 8a 4b, 5, 6, 9
6.75 d (9.1) 6.93 d (9.1)
4b, 5, 6 4b, 5, 6
3.48 d (7.2) 5.27 t (7.2)
1, 2, 3, 20 , 30 1 0 , 40 , 50
3.40 d (7.1) 5.17 t (7.1)
2, 3, 20 , 30 10 , 40 , 50
1.86 s 1.78 s 2.91 d (7.2) 2.51 quint (7.2)
20 , 30 , 50 20 , 30 ,40 3, 4, 4a, 200 , 300 , 1000 4, 4a, 100 , 400 , 500 , 1000 , 2000
1.81 s 1.66 s 3.00 d (7.4) 2.51quint (7.4)
20 , 30 , 50 20 , 30 ,40 3, 4a, 200 , 300 , 400 , 1000 100 , 300 , 400 , 500 , 1000 , 2000
4.68, 4.64 br s 1.75 s 2.19, 2.18 t (7.1) 5.13 t (7.1)
200 , 500 4a, 200 , 400 4a, 100 , 200 , 2000 , 3000 200 , 1000 , 4000 , 5000
4.78, 4.71 br s 1.75 s 2.14 t (6.9) 5.04 t (6.9)
200 ,500 200 , 300 , 400 , 100 , 200 , 300 , 2000 , 3000 200 , 1000 , 4000 , 5000
1.58 s 1.68 s 13.31 s 6.28 s 5.63 s
2000 , 3000 , 5000 2000 , 3000 , 4000 1, 2, 9a 2, 3, 4 4b, 5, 6
1.56 s 1.66 s 11.08 s 6.27 s e 5.53 s 3.99 s
2000 ,3000 ,5000 2000 ,3000 ,4000 1, 2, 9a 2, 3, 4 e 5, 6,7 5
4.04 s
6
Determined by HSQC and HMBC correlations.
subjected to further purification by RP18-HPLC (Agilent XDB-C18 PrepHT; flow rate 1 mL/min) using a MeOH (50%)-2-propanol gradient (35% 2-propanol 0e18 min; 35% / 50% 2-propanol 18e21 min) and yielded compounds 7 (7.75 mg, tR 0.28) and 8 (29.23 mg, tR 0.29). The MeOH extract (29.16 g) was diluted to water and extracted with EtOAc and butanol, successively. Based on the TLC results, the EtOAc extract (8.26 g) was selected for further fractionation. Part of this extract (734.1 mg) was purified over column chromatography on Sephadex LH-20 (30 4 cm) eluted with MeOH 100% (flow rate 0.5 mL/min) and yielded 210 fractions (z10 mL each), which were combined based on TLC similarities to 14 fractions (KEM.A.Se1KEM.A.Se14), three of which constituted compounds 10 (32.4 mg, fraction KEM.A.Se7), 9 (17.6 mg, fraction KEM.A.Se12). 11 (6.6 mg, fraction KEM.A.Se13) and 12 (KEM.A.Se14, 7.1 mg). Furthermore KEM.A.Se1 (155.2 mg) was applied to column chromatography over silica gel (8.5 2.5 cm), eluted with mixtures of increasing polarity of DCM-MeOH-H2O (flow rate 2 mL/min) and yielded compound 13 (9.4 mg, DCM-MeOH-H2O 76:24:2.4). 3.4.1. Kellerine A (1,2-dihydro-3,8-dihydroxy-6-methoxy-1,1,5tri(3-methylbut-2-enyl)xanthen-2,9-dione) (1) Yellowish Oil; UV (MeOH) lmax 300 nm (log ε 3.5); 1H and 13C NMR data, see Tables 1 and 2; HRESIMS: [М-Н]- at m/z: 477.2289. C29H33O6 requires 477.2283.
data, see Tables 1 and 2; HRESIMS: [М-Н]- at m/z: 463.2139. C28H31O6 requires 463.2126. 3.4.3. 6-Methylpatulone (1,2-dihydro-3,8-dihydroxy-6-methoxy1,1-bi (3-methylbut-2-enyl)xanthen-2,9-dione) (3) Yellowish oil; UV (MeOH) lmax 310 nm (log ε 3.4); 1H and 13C NMR data, see Tables 1 and 2; HRESIMS: [М-Н]- at m/z: 409.1662. C24H25O6 requires 409.1657. 3.4.4. (200 R/S)-kellerine C ((R/S)-1,3,5-trihydroxy-2-(3-methyl-2buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3-enyl]-6methoxy-9H-xanthen-9-one) (4) Yellowish oil; ½a25 D þ8 (c 0.1); UV (MeOH) lmax 250 nm (log ε 4.6), 330 nm (log ε 4.3); 1H and 13C NMR data, see Table 3; CDspectrum see supplementary material; HRESIMS: [М-Н]- at m/z: 477.2286. C29H33O6 requires 477.2283. 3.4.5. (200 R/S)-creticine ((R/S)-1,3,6-trihydroxy-5-methoxy-2-(3methyl-2-buten-1-yl)-4-[2-(3-methylbut-2-enyl)-3-methylbut-3enyl]-11Н-dibenzo[b,e][1,4]dioxepin-9-one) (5) Brownish-orange oil; ½a25 D -6 (c 0.2, MeOH); UV (MeOH) lmax 1 13 (log ε) 270 (4.0); H and C NMR data, see Table 3; CD-spectrum see supplementary material; HRESIMS: [М-Н]- at m/z: 493.2236. C29H33O7 requires 493.2232. Acknowledgements
3.4.2. Kellerine B (1,2-dihydro-3,6,8-trihydroxy-1,1,5-tri(3methylbut-2-enyl)xanthen-2,9-dione) (2) Yellowish Oil; UV (MeOH) lmax 300 (log ε 3.7); 1H and 13C NMR
We thank F. Kastner, A. Schramm and G. Stühler for measuring the NMR (Bruker Avance III 600 kryo and Bruker Avance III HD 400),
A. Mathioudaki et al. / Phytochemistry 146 (2018) 1e7
€llner for recording the MS spectra (all as well as J. Kiermeier and So Central Analytics of NWF IV, University of Regensburg). PD Dr. A. Dürkop (Institute of Analytical Chemistry of NWF IV, University of Regensburg), J. Ziegler and S. Wiesneth (both Institute of Pharmaceutical Biology of NWF IV, University of Regensburg) are acknowledged for performing the CD experiments. We are indebted to Ms. Gabi Brunner for excellent technical assistance and performing the cytotoxicity assays. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.phytochem.2017.11.009. References Athanasas, K., Magiatis, P., Fokialakis, N., Skaltsounis, A.L., Pratsinis, H., Kletsas, D., 2004. Hyperjovinols A and B: two new phloroglucinol derivatives from Hypericum jovis with antioxidant activity in cell cultures. J. Nat. Prod. 67, 973e977. Avato, P., 2005. A survey of the Hypericum genus: secondary metabolites and bioactivity. Stud. Nat. Prod. Chem. 30, 603e634. Azebaze, A.G.B., Meyer, M., Valentin, A., Nguemfo, E.L., Fomum, Z.T., Nkengfack, A.E., 2006. Prenylated xanthone derivatives with antiplasmodial activity from Allanblackia monticola Staner L.C. Chem. Pharm. Bull. 54, 111e113. Battistini, I., 2013. Analisi LC-PDA-MS di estratti di specie di Hypericum raccolte in Tunisia: studio fitochimico dell',estratto metanolico di Hypericum humifusum L. (Hypericaceae), 2013, Tesi di laurea specialistica LC5, URN etd12182012144131. Chang, S.W., Kim, H.K., Lee, I.K., Sang, U.C., Ryu, S.Y., Kang, R.L., 2009. Phytochemical constituents of Bistorta manshuriensis. Nat. Prod. Sci. 15, 234e240. Crockett, S.L., Robson, N.K.B., 2011. Taxonomy and chemotaxonomy of the genus Hypericum. Med. Arom. Plant. Sci. Biotechnol. 5, 1e13. Crockett, S.L., Wenzig, E.-M., Kunert, O., Bauer, R., 2008. Anti-inflammatory phloroglucinol derivatives from Hypericum empetrifolium. Phytochem. Lett. 1, 37e43. Ernst, E., 2003. The Genus Hypericum L. CRC Press. Farinha, A., Martins, J.M., Nogueira, T., Tavares, R., Duarte, F.A., 2002. HPLC analysis of Hypericum L. Species from Portugal, natural products in the new millennium: prospects and industrial application. In: Volume 47 of the Series Proceedings of the Phytochemical Society of Europe, pp. 125e134. Heilmann, J., Wasescha, M.R., Schmidt, T.J., 2001. The influence of glutathione and cysteine levels on the cytotoxicity of helenanolide type sesquiterpene lactones against KB cells. Bioorg. Med. Chem. 9, 2189e2194. Hyun, S.K., Jung, H.A., Min, B.-S., Jung, J.H., Choi, J.S., 2010. Isolation of phenolics, nucleosides, saccharides and an alkaloid from the root of Aralia cordata. Nat. Prod. Sci. 16, 20e25. Ishiguro, K., Nagareya, N., Suitani, A., Fukumoto, H., 1997. A prenylated xanthone from cell suspension cultures of Hypericum patulum. Phyto¢hemistry 44, 1065e1066. Ito, C., Itoigawa, M., Mishina, Y., Tomiyasu, H., Litaudon, M., Cosson, J.-P., Mukainaka, T., Tokuda, H., Nishino, H., Furukawa, H., 2001. Cancer chemopreventive agents. New depsidones from Garcinia plants. J. Nat. Prod. 64, 147e150. Karioti, A., Vincieri, F.F., Bilia, A.R., 2009. Rapid and efficient purification of naphthodianthrones from St. John's wort extract by using liquideliquid extraction and SEC. J. Sep. Sci. 32, 1374e1382. Karioti, A., Bilia, A.R., 2010. Hypericins as potential leads for new therapeutics. Int. J. Mol. Sci. 11, 562e594.
7
Kitanov, G.M., Nedialkov, P.T., 1998. Mangiferin and isomangiferin in some Hypericum species. Biochem. Syst. Ecol. 26, 647e653. Lebreton, P., Bouchez, M.-P., 1967. Recherches chimiotaxonomiques sur les plantes te rosides polyphe noliques du genre Hypericum. Plant. Med. vasculaires. 9. Les he Phytother. 1, 188e200. Mabry, T.G., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavonoids. Springer-Verlag, Berlin, Heidelberg, New York. Makovetska, O.Y., 2000. Research on biologically active substances of Hypericum L. species. Report VII. Sections Drosocarpium Spach., Oligostema (Boiss.) Stef., Thasia Boiss. and Crossophyllum Spach. Farmatsevtichnii Zhurnal (Kiev) 3, 53e59. Neu, R., 1957. Chelate von Diarylbors€ auren mit aliphatischen Oxyalkylaminen als Reagenzien für den Nachweis von Oxyphenyl-benzo-g-pyronen. Die Naturwiss. 44, 181. Ngoupayo, J., Tabopda, T.K., Muhammad, S.A., Tsamo, E., 2008. a-Glucosidase inhibitors from Garcinia brevipedicellata (Clusiaceae). Chem. Pharm. Bull. 56, 1466e1469. Nürk, N.M., Crockett, S.L., 2011. Morphological and phytochemical diversity among Hypericum species of the Mediterranean Basin. Med. Aromatic Plant Sci. Biotechnol. 5 (Special Issue 1), 14e28. Permanaa, D., Abas, F., Maulidiani, Shaari K., Stanslas, J., Ali, A.M., Lajis, N.H., 2005. Atrovirisidone B, a new prenylated depsidone with cytotoxic property from the roots of Garcinia atroviridis. Z. Naturforsch. 60c, 523e526. Rios, M.Y., Delgado, G., 1992. Polyprenols and acylphloroglucinols from Esenbeckia nesiotica. Phytochemistry 31, 3491e3494. Robson, N.K.B., 2010. Studies in the genus Hypericum L. (Hypericaceae) 5(1). Sections 10. Olympia to 15/16. Crossophyllum. Phytotaxa 4, 5e126. Rouis, Z., Abid, N., Aouni, M., Faiella, L., Dal Piaz, F., De Tommasi, N., Braca, A., 2013. Benzophenone glycosides from Hypericum humifusum ssp. australe. J. Nat. Prod. 76, 979e982. Schmidt, S., Jürgenliemk, G., Skaltsa, H., Heilmann, J., 2012a. Phloroglucinol derivatives from Hypericum empetrifolium with antiproliferative activity on endothelial cells. Phytochemistry 77, 218e225. Schmidt, S., Jürgenliemk, G., Schmidt, T.J., Skaltsa, H., Heilmann, J., 2012b. Bi-, tri-, and polycyclic acylphloroglucinols from Hypericum empetrifolium. J. Nat. Prod. 75, 1697e1705. Seabra, R.M., Alves, A.C., 1989. Mangiferin and chlorogenic acid from Hypericum species. Planta Medica 55, 404. Tanaka, N., Mamemura, T., Abe, S., Imabayashi, K., Kashiwada, Y., Takaishi, Y., Suzuki, T., Takebe, Y., Kubota, T., Kobayashi, J., 2010. Biyouxanthones A - D, prenylated xanthones from roots of Hypericum chinense. Heterocycles 80, 613e621. Tatsis, E.C., Boeren, S., Exarchou, V., Troganis, A.N., Vervoort, J., Gerothanassis, I.P., 2007. Identification of the major constituents of Hypericum perforatum by LC/ SPE/NMR and LC/MS. Phytochemistry 68, 383e393. Tatsis, E.C., Exarchou, V., Troganis, A.N., Gerothanassis, I.P., 2009. 1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups. Anal. Chim. Acta 607, 219e226. Trigas, P., Panitsa, M., Tsiftsis, S., 2013. Elevational gradient of vascular plant species richness and endemism in Crete e the effect of post-isolation mountain uplift on a continental island system. PLoS ONE 8, e59425. Umek, A., Kreft, S., Kartnig, T., Heydel, B., 1999. Quantitative phytochemical analyses of six Hypericum species growing in Slovenia. Planta Med. 65, 388e390. Winkelmann, K., San, M., Kypriotakis, Z., Skaltsa, H., Bosilij, B., Heilmann, J., 2003. Antibacterial and cytotoxic activity of prenylated bicyclic acylphloroglucinol derivatives from Hypericum amblycalyx. Z. Naturforsch. 58c, 527e532. Xu, Y.-J., Chiang, P.-Y., Lai, Y.-H., Vittal, J.J., Wu, X.-H., Tan, B.K.H., Imiyabir, Z., Goh, S.H., 2000. Cytotoxic prenylated depsidones from Garcinia parvifolia. J. Nat. Prod. 63, 1361e1363. Xu, W.-J., Li, R.-J., Quasie, O., Yang, M.-H., Kong, L.-Y., Luo, J., 2016. Polyprenylated tetraoxygenated xanthones from the roots of Hypericum monogynum and their neuroprotective activities. J. Nat. Prod. 79, 1971e1981.