Phytochemistry xxx (2013) xxx–xxx
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Cancer chemopreventive diterpenes from Salvia corrugata Emanuela Giacomelli a,1, Samuel Bertrand b,1, Andreas Nievergelt b, Vincent Zwick b, Claudia Simoes-Pires b, Laurence Marcourt b, Elisabeth Rivara-Minten b, Muriel Cuendet b, Angela Bisio a, Jean-Luc Wolfender b,⇑ a b
Department of Pharmacy, University of Genoa, via Brigata Salerno 13, 16147 Genoa, Italy School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
a r t i c l e
i n f o
Article history: Received 24 June 2013 Received in revised form 7 September 2013 Available online xxxx Keywords: Salvia corrugata Diterpenes Abietane Icetexane Fruticulin C Quinone reductase Histone deacetylase
a b s t r a c t NMR and NP-HPLC-UV profiling of the exudate of Salvia corrugata revealed that its secondary metabolite composition was largely dominated by a-hydroxy-b-isopropyl-benzoquinone diterpenoids. Among them, four diterpenes not described previously were isolated and identified as fruticulin C (3), 7a-methoxy-19acetoxy-royleanone (4), 7a,19-diacetoxy-royleanone (5), and 7-dehydroxy-conacytone (7). In addition, the known diterpenes fruticulin A (1), demethyl-fruticulin A (2) and 7a-O-methyl-conacytone (6) were also obtained. The isolated compounds were evaluated for their cancer chemopreventive activity by measuring quinone reductase induction activity and histone deacetylase inhibition. Three compounds (1, 2 and 5) showed promising activity. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The main secondary metabolites of Salvia species (Lamiaceae family) are terpenoids and flavonoids (Topçu, 2006). Among terpenoids, diterpenes are the largest group, classified into subgroups, such as abietane, clerodane, pimarane, labdane (Wu et al., 2012). Abietanes and rearranged abietanes comprise the largest group of known Salvia diterpenoids (Wu et al., 2012). Abietanes from Salvia species are known to possess various biological activities such as antibacterial, antiprotozoal, antioxidant, anti-inflammatory, antitumor, antihypertensive, as well as phytotoxic and DNA damaging properties (Ebrahimi et al., 2013; Topçu and Ahmet, 2007; Wu et al., 2012). A sub-class of abietanes, namely icetexanes, has shown antimicrobial (El-Lakany et al., 1995), insecticidal (Fraga et al., 2005), trypanocidal (Sanchez et al., 2006; Uchiyama et al., 2003) and anticancer activities (Fronza et al., 2011, 2012).
⇑ Corresponding author. Address: Phytochimie et Produits Naturels Bioactifs, Ecole de Pharmacie Genève-Lausanne, Section des Sciences Pharmaceutiques, Université de Genève, Quai Ansermet 30, CH-1211 Genève 4, Switzerland. Tel.: +41 22 379 33 85; fax: +41 22 379 33 99. E-mail address:
[email protected] (J.-L. Wolfender). 1 E. Giacomelli and S. Bertrand equally contributed to this work.
Salvia corrugata Vahl. is an American species (Epling, 1939) widely cultivated for ornamental purposes. Two main icetexane diterpenoids, namely fruticulin A (1) and demethyl-fruticulin A (2), were already isolated and showed bactericidal and bacteriostatic activities (Bisio et al., 2008; Schito et al., 2011). Moreover, demethyl-fruticulin A (2) was found to possess antitumor activity in cell cultures (Giannoni et al., 2010) and to induce phase II metabolising enzymes (Monticone et al., 2010). From a chemical point of view, these icetexane compounds have a ring C with an a-hydroxy-b-isopropyl-benzoquinones moiety, which could be of interest for an interaction with phase II metabolising enzymes (Bensasson et al., 2008). In the present study, the exudate of S. corrugata was submitted to an in depth phytochemical investigation to obtain more comprehensive information on its abietane diterpene composition and to search for novel a,b-unsaturated ketones that were investigated for their bioactivity. All constituents were identified by 1D and 2D NMR spectroscopy, as well as HR-MS analysis. Because of previously reported activities, the exudate was evaluated for its cancer chemopreventive properties through bioassays targeting various stages of carcinogenesis. Induction of quinone reductase (QR), a phase II metabolising enzyme, as well as histone deacetylase (HDAC) inhibition were found. The activity of the isolated compounds against these two targets was then measured.
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.09.011
Please cite this article in press as: Giacomelli, E., et al. Cancer chemopreventive diterpenes from Salvia corrugata. Phytochemistry (2013), http://dx.doi.org/ 10.1016/j.phytochem.2013.09.011
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The 1H-NMR spectrum of the exudate (Fig. 1B) showed that, in addition to the typical signals of methyl and methylene groups (between dH 0.7 and 1.2) most probably related to triterpenoids (Topçu and Ulubelen, 1999), the main constituents were fruticulin A (1) and demethyl-fruticulin A (2) (Fig. 1A) (Bisio et al., 2008). They were both identified by their downfield aromatic proton H-20 (dH 8.15 for 1 and 8.07 for 2), the aromatic multiplet between dH 7.0 and 6.75 (H-1, 3 and 7), the methylene doublet at dH 3.12 (H-6), the isoprenyl chain with its characteristic methine septuplet at dH 3.37 (H-15), and the two methyl group at dH 1.26 (H-16 and H-17).
2. Results and discussion 2.1. NMR and NP-HPLC profiling of S. corrugata exudate The chemical diversity of the non-polar constituents present in the exudate material of S. corrugata, which was obtained by brief dipping in dichloromethane (Bisio et al., 2008), was assessed by NMR (Al-Massarani et al., 2012; Wolfender et al., 2013) and normal-phase (NP) HPLC-PDA profiling (Fig. 1).
A 3.5 8.0
7.0
3.0
6.0
5.0
(ppm)
4.0
3.0
2.0
1.0
B 3.5
3.0 1 3 4
(ppm)
2
5 6
C
7 20 30 40 50 60
D
70 8.0
100
6.0
5.0
E
(ppm)
4.0
3.0
2.0
80 60
3
60
40
2
40 20
3000
80 100
1
80
4000
1.0
4
7
5
20
6
0
1
F
% EtOAc
120
7.0
2
2000 1000 4 5 7
0
10
3 6
20
30
40
60
QR Induction Zone Fig. 1. NMR (CDCl3) and NP-HPLC profiling (column: Cosmosil 5Si-II 4.6 250 mm 5 lm, flow rate: 1.0 ml/min, an isocratic step at 5% ethyl acetate in n-hexane for 5 min followed by a linear gradient to 100% ethyl acetate in 60 min; see section 4.4 for more details) of the dichloromethane crude exudate from S. corrugata. (A) 1H-NMR spectrum of demethyl-fruticulin A; (B) 1H-NMR spectrum of the exudate for comparison with the spectrum of demethyl-fruticulin A, (C) gCOSY NMR spectrum of the exudate, (D) gHSQC NMR spectrum of the exudate; (E) HPLC-UV profile of the exudate at 450 nm; (F) HPLC-ELSD profiles of exudate. The ‘‘QR induction zone’’ indicates the chromatographic zone where the related bioactive compounds are eluting.
Please cite this article in press as: Giacomelli, E., et al. Cancer chemopreventive diterpenes from Salvia corrugata. Phytochemistry (2013), http://dx.doi.org/ 10.1016/j.phytochem.2013.09.011
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E. Giacomelli et al. / Phytochemistry xxx (2013) xxx–xxx
The gHSQC spectrum of the exudate showed a second similar correlation between dH 3.18 and dC 24.5 close to the methine correlation of the isoprenyl group. The COSY correlations of this typical methine group with the methyl groups at dH 1.20 confirmed the presence of an isopropyl group most probably related to diterpene(s) containing an a-hydroxy-b-isopropyl-benzoquinone moiety. An extensive literature search indicated that more than 30 a-hydroxy-b-isopropyl-benzoquinone have already been reported in the genus Salvia either with icetexane or abietane skeletons (Supplementary material I) (Cardenas and Rodriguez-Hahn, 1995; Esquivel et al., 1997; Frontana et al., 1997; Frontana and Gonzalez, 2007; Galicia et al., 1988; Nieto et al., 2000; Ortega et al., 1995; Ulubelen et al., 1992). In the case of abietane diterpenes, the methine proton of the isopropyl group is slightly shifted upfield to dH 3.18 compared to icetexanes (Cardenas and Rodriguez-Hahn, 1995; Galicia et al., 1988; Hernandez et al., 1987; Nieto et al., 2000; Ortega et al., 1995; Ulubelen et al., 1992). Therefore, the NMR profile indicated that, in addition to the known icetexanes, abietane diterpenoid benzoquinones were also present in the exudate. To obtain a comprehensive view of all a-hydroxy-b-isopropylbenzoquinones present in the exudate, an HPLC profiling was performed. As the dichloromethane exudate mainly contains non-polar constituents, it was profiled by NP-HPLC coupled to both a photodiode array (PDA) and an evaporative light scattering detector (ELSD) (Wolfender, 2009). Since a-hydroxy-b-isopropyl-benzoquinones are reported to exhibit orange to yellow colours (Supplementary material I) (Cardenas and Rodriguez-Hahn, 1995; Esquivel et al., 1997; Jimenez et al., 1988; Nieto et al., 2000; Ulubelen et al., 1992), the PDA trace recorded at 450 nm (Fig. 1E) was used to monitor more specifically these constituents which were also detected at 270 nm. The chromatogram showed the presence of at least 17 constituents probably sharing the same chromophore (Fig. 1E). The ELSD trace (Fig. 1F) revealed the presence of two additional main constituents which were attributed to the previously isolated triterpenes (Bisio et al., 2008). This detection mode also confirmed that the two main diterpenes were fruticulin A (1) and demethyl-fruticulin A (2). As quinones were previously reported to exhibit promising QR inducing activities (Deng et al., 2007; Misico et al., 2002; Pawlus et al., 2005) and because of our interest in the search for new cancer chemopreventive compounds, the presence of such compounds in the exudate of S. corrugata led us to explore this activity. The exudate exhibited a promising induction ratio (IR) of 4.3 at 20 lg/ml, and the profiling results revealed the presence of a-hydroxy-b-isopropyl-benzoquinones not yet reported in this Salvia species. Given this, a bioassay-guided isolation of the active constituents of S. corrugata was undertaken.
semi-preparative procedures were obtained by geometric chromatographic scale–up (Guillarme et al., 2008, 2009). Repeated semi-preparative HPLC fractionation led to the purification of 7 constituents. 2.3. Identification of quinones from S. corrugata Each constituent (Fig. 2) was identified by spectroscopic NMR analyses, including 1H, APT, gCOSY, gHSQC, gHMBC and NOESY experiments, and its structure confirmed by HR-MS analysis. Tables 1 and 2 contain NMR data of compounds 3, 4, 5 and 7. The 1H-NMR spectra of each isolated constituent showed the typical septuplet of the isopropyl methines of a-hydroxy-b-isopropyl-benzoquinones at either dH 3.17 or dH 3.35 (H-15), as already highlighted in the NMR profile of the exudate. The two methyl of the isopropyl group were observed either as single doublets (6H) for planar structures such as fruticulin A (1) or as two separate doublets for structures containing asymmetric carbons such as in abietane skeletons. The HMBC correlations between the two aromatic carbons C-12 and C-13 and the carbonyl C-14 with both H15 and the phenolic proton (OH-12) confirmed the presence of an a-hydroxy-b-isopropyl-benzoquinone (C ring) for each isolated diterpenoid (Topcu and Ulubelen, 2007). Compounds 1 and 2 were identified as the two icetexanes fruticulin A and demethyl-fruticulin A (Bisio et al., 2008) and 6 as the abietane 7a-O-methyl-conacytone from S. candicans (Cardenas and Rodriguez-Hahn, 1995) on the basis of their 1H-NMR spectra. The other a-hydroxy-b-isopropyl-benzoquinones 3, 4, 5 and 7 could not be readily identified based on the literature. Compound 3 was obtained as a red amorphous powder. The HR ESI-TOF/MS of 3 in negative ion mode (NI) displayed a [MH] at m/z 353.1382. This suggested a molecular weight of 354.1467. The corresponding molecular formula was C21H22O5. In addition to the characteristic signals of an a-hydroxy-b-isopropyl-benzoquinone [dH 3.23 (1H, sept, J = 7.1 Hz, H-15), 1.23 (3H, d, J = 7.1 Hz, H-17), 1.21 (3H, d, J = 7.1 Hz, H-16)], the 1H-NMR spectrum of 3 consisted of two meta-coupled aromatic protons (H-3 and H-1), a methoxyl group (H-19) and a methyl group (H-18). These signals showed a close similarity with those from the A ring of the icetexanes 1 and 2. The remaining signals related to the B ring consisted of two ortho aromatic protons (H-6 and H-7) and a highly downfield shifted methine singlet at dH 5.81 (H-20). This latter signal was attributed to a carbon at dC 74.6 (C-20) having an HMBC correlation with a methoxyl group (H-21). The 3JCH HMBC correlations of the B ring protons (H-20 with C-1 (A ring); H-20 with C-11 (C-ring); H-6
20 1
2.2. Bioassay-guided isolation of constituents from S. corrugata The exudate of S. corrugata aerial parts was subjected to an initial fractionation by High Speed Counter Current Chromatography (HSCCC) in order to ensure a full recovery of the sample injected and avoid possible bioactivity loss. The Arizona solvent systems (Berthod et al., 2005) were tested. The partition coefficient of the major compounds was estimated after HPTLC profiling of each phase. The appropriate solvent system (P) was finally selected since it provided the best repartition of the compounds of interest between the two phases. Nine fractions (P1-P9) were obtained by HSCCC fractionation and further analysed by NP-HPLC. Fractions with the higher QR induction activity and with complementary chemical composition (P4, P7 and P9) were selected for further purification of a-hydroxy-b-isopropyl-benzoquinones. Final purification was achieved by NP-HPLC. For this, the analytical conditions were optimised for each fraction and the
OH
O
O R
2
10
A
9
11
C 14
B 5
16 21
15
8
13
OH
O
O
17
O
O
O
6
19
18
1: R=CH3 (fruticulin A) 2: R=H (demethyl fruticulin A) OH
O 2
20
1
11 9 14
10 22
O
16 15
HO O
OH
20 17
O
8
O
21
13
3 (fruticulin C)
5 19
7
18
4: R=CH3 5: R=Ac
O O R
O 19
R 6: R=OCH3 7: R=H
Fig. 2. Isolated diterpenes from the exudate of S. corrugata.
Please cite this article in press as: Giacomelli, E., et al. Cancer chemopreventive diterpenes from Salvia corrugata. Phytochemistry (2013), http://dx.doi.org/ 10.1016/j.phytochem.2013.09.011
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Table 1 H NMR spectral data for compounds 3, 4, 5, and 7 (500 MHz, CDCl3).
1
H
*
3 (J in Hz)
4
5
7
1
6.86 d (2.6)
2
–
3
6.87 d (2.6)
5 6
– 7.75 d (12.3)
(equ.)2.71 brd (13.1) (ax.)1.22 m (ax.)1.71 m, (equ.)1.57 m (equ.)1.72 brd (14.3) (ax.)1.15 td (14.3, 4.2) (ax.)1.75 dd (13.0, 1.5) (equ.) 2.17 ddd (14.3, 1.9, 1.5) (ax.) 1.42 ddd (14.3, 13.0, 3.3) – 4.31 dd (3.3, 1.9) 7.11 s 3.18 sept (7.2) 1.20 d (7.2) 1.23 d (7.2) 1.03 s 4.18 d (11.2) 4.03 d (11.2) 1.22 s – – 2.08 s 3.46 s –
2.76 brd (12.6) 1.26 m 1.72 m 1.60 m 1.71 m 1.16 m 1.64 m 2.10 m 1.68 m – 5.93 dd (3.4, 1.7) 7.12 s 3.16 sept (7.0) 1.19 d (7.0) 1.23 d (7.0) 0.97 s 4.15 d (11.2) 4.00 d (11.2) 1.25 s – – 2.06 s – 2.04 s
2.60 dd (13.3, 6.1) 1.25 m 2.43 qt (13.3, 6.1) 1.58 dt (13.3, 6.1) 1.75 dd (13.3, 6.1) 1.40 tdd (13.3, 6.1, 2.7) 1.23 m 1.80 m 2.07 dtd (12.8, 11.6, 5.2) 2.26 ddd (20.5, 11.6, 6.6) 2.85 ddd (20.5, 5.2, 1.1) 7.15 s 3.18 sept (7.1) 1.21 d (7.1) 1.21 d (7.1) 0.80 s 3.92 dd (11.2, 2.7)* 3.33 dd (11.2, 1.2)* 5.63 d (2.7) 2.12 d (2.7) – – – –
7 (ax.) 7 (equ.) OH (12) 15 16 17 18 19
7.33 – 7.24 3.23 1.21 1.23 2.51 3.87
d (12.3)
20 OH (20) 21 22 23 24
5.81 s – 3.02 s – – –
s sept (7.1) d (7.1) d (7.1) s s
The second J-values come from long-range coupling (‘‘W-coupling’’, 4JH19a–H3ax and 4JH19b–H5ax), as confirmed by the COSY correlations.
Table 2 C NMR spectral data for the compounds 3, 4, 5, and 7 (125 MHz, CDCl3).
13
C
3
4
5
7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
111.8 162.2 117.7 140.5 126.7 137.3 120.3 137.6 127.5 137.6 182.9 151.2 125.3 187.1 24.6 20.0 20.0 21.2 55.6 74.6 56.0 – – –
35.8 18.7 36.1 36.8 46.1 22.7 70.79 141.4 147.44 39.0 184.2 150.8 125.0 186.4 24.4 19.9 20.0 27.4 67.7 18.8 171.4 21.1 57.6 –
36.0 18.7 36.4 36.7 46.7 25.3 64.5 139.5 149.5 39.0 183.8 151.0 125.0 ND 24.3 19.8 20.0 27.6 67.8 18.7 171.3 21.1 169.8 21.2
35.3 21.1 40.2 32.9 46.6 18.0 25.5 148.0 140.7 41.0 183.6 150.5 124.5 187.5 24.1 19.8 19.8 23.8 66.1 95.1 – – – –
ND: not detected.
with C-4 (A ring); H-7 with C-14 (C ring)) allowed to fully characterise the B ring and to identify 3. Its dioxoicetexane structure is very similar to that of fruticulin A (1) and B. To our knowledge, this constituent has not yet been reported and the name fruticulin C is therefore proposed. Due to the restricted amount of 3, the stereochemistry of C-20 could not be determined. Compound 4 was obtained as a yellow amorphous powder ([MH] at m/z 403.2168, corresponding to the molecular formula C23H32O6). The presence of the septuplet at dH 3.18 and the two doublets at dH 1.20 and 1.23 in the 1H-NMR spectrum of 4 indicated a six-membered B ring attached to the p-benzoquinone C ring, which is typical for an abietane a-hydroxy-b-isopropylbenzoquinone (Topcu and Ulubelen, 2007). Additionally, the HSQC
spectrum indicated the presence of two methyl groups (dC 18.8 and 27.4), an acetate (dC 21.1), three oxygenated groups (a methine at dC 70.8, a methylene at dC 67.7 and a methyl at dC 57.6), four methylenes and a methine. This methyl (dC 18.8) was localised at position C-10 because of the HMBC correlations between the methyl protons (H-20) at dH 1.22 and the aromatic carbon at dC 147.4 (C-9). Furthermore, the methylene protons (H2–19) were correlated with the methyl at dC 27.4 (C-18), the methine at dC 46.1 (C-5), the methylene at dC 36.1 (C-3) and the quaternary carbon at dC 46.1 (C-4) which proved its attachment to the C-4 position of the A ring. These methylene protons (H2–19) were also linked to an acetyl (HMBC correlation of dC 171.4 (C-21) with both H-19 and the methyl H-22 at dH 2.08). The methoxy group was positioned in C-7 (3JC–H correlation: dH 3.46 with dC 70.8 (C-7)) and the small coupling constant of H-7 with H-6 ax. (J = 3.3 Hz) proved its axial position. The NOESY correlation between H-6 ax, H-19 and H-20 on one side, and between H-6 eq, H-5 and H-18 on the other, allowed the identification of 4 as 7a-methoxy-19-acetoxy-royleanone (7a-methoxy-12-hydroxy-19-acetoxy-11,14-dioxoabieta8,12-diene). To our knowledge, this compound is a new abietane diterpene, and its NMR spectra were in good agreement with compounds sharing the same skeleton (Cardenas and Rodriguez-Hahn, 1995; Nieto et al., 2000). Compound 5 was obtained as a yellow/orange amorphous powder ([M-H]- at m/z 431.1939, corresponding to the molecular formula C24H32O7). It showed 1H- and 13C-NMR spectra similar to those of 4 (Tables 1 and 2). The only difference was the lack of the methoxy group and an additional methyl singlet at dH 2.04 (H-24). This signal was due to an acetoxy group at C-7 as shown by the HMBC correlations between the carbonyl C-23 (dC 169.8) with H-24 (dH 2.04) and H-7 (dH 5.93). Compound 5 was thus identified as 7a,19-diacetoxy-royleanone (7a,19-diacetoxy-12-hydroxy-11,14-dioxoabieta-8,12-diene). Compound 7 was isolated as a yellow powder ([M-H]- at m/z 345.1661, corresponding to the molecular formula C20H26O5). The NMR spectra of 7 (Tables 1 and 2) showed high similarities with those of 7a-O-methyl-conacytone (6) (Cardenas and RodriguezHahn, 1995). The differences only consisted in the lack of both the methoxy group and the oxygenated methine proton (H-7)
Please cite this article in press as: Giacomelli, E., et al. Cancer chemopreventive diterpenes from Salvia corrugata. Phytochemistry (2013), http://dx.doi.org/ 10.1016/j.phytochem.2013.09.011
E. Giacomelli et al. / Phytochemistry xxx (2013) xxx–xxx
and the presence of a methylene at dH 2.85 and 2.36 (H-7). This indicated the absence of substitution at C-7. Therefore, 7 was identified as 7-dehydroxy-conacytone. The dipolar correlations between H-20 (dH 5.63) and H-1 eq (dH 2.60) confirmed that 7 shares the same configuration as conacytone. To our knowledge, compounds 3, 4, 5, and 7 have not yet been reported and are presented here for the first time. 2.4. Quinone reductase induction of constituents from S. corrugata Quinones and other a,b-unsaturated ketones are known inducers of phase II metabolising enzymes (Bensasson et al., 2008). These enzymes are thought to protect from the development of cancer by inactivating reactive metabolites and xenobiotics. Phase II enzyme expression is under the control of two distinct pathways; arylhydrocarbon receptor (AhR) mediated activation of the xenobiotic response element and Keap1/Nrf2 mediated activation of the antioxidant response element (ARE) (Fahey et al., 2004). When both pathways are involved, QR inducers are considered to be bifunctional and are of less interest for cancer chemoprevention due to their possible effects on P450 enzymes. Compounds leading to the activation of ARE only are considered to be monofunctional inducers and are regarded as beneficial for chemoprevention (Köhle and Bock, 2007). The enzymatic induction of the isolated constituents from S. corrugata (Table 3) was measured in two distinct cell lines, namely Hepa1c1c7 and its clone c35. This allowed the distinction between mono- and bifunctional inducers as the former cell line possesses functional genes for both pathways whereas the latter expresses a non-functional AhR mutant being therefore only susceptible to ARE mediated enzyme induction. The isolated constituents of S. corrugata showed a comparatively good monofunctional induction of QR enzyme with a concentration to double its expression (CD value) in the low lM range for both cell lines (Table 3). Apart from 5, they exhibited only a moderate in vitro toxicity against the used cell lines (Table 3). As positive standards, 40 -bromoflavone for the Hepa1c1c7 and isoliquiritigenin for the c35 cell line were used, exhibiting CD values of 23.0 nM and 6.8 lM, respectively and no apparent toxicity at the used concentrations. To date, most QR inducers of natural origin have not shown any activity below the lM range (Kang and Pezzuto, 2004; Talalay et al., 1988). Their mode of action generally relies on a nucleophilic addition by Keap1 cysteine residues (Michael reaction) (Fahey et al., 2004) or an oxidative stress response caused by redox-cycling (Erlank et al., 2011). The underlying molecular mechanism remains to be elucidated. For 1 and 2 a nucleophilic addition of a cysteine at carbon 7 of the a,b-unsaturated ketone moieties may be hypothesised. The activity loss of 4 compared to 5 may be due
Table 3 QR induction of selected isolated diterpenes. Compounds
1 2 3 4 5 6 7 40 -bromoflavone* Isoliquiritigenin* *
Hepa1c1c7
c35
CD [lM]
IC50 [lM]
CI
CD [lM]
IC50 [lM]
CI
1.4 3.1 7.6 19.6 1.5 14.0 11.2 0.03 7.3
37.2 17.6 16.3 >49.4 8.7 50.1 59.5 >0.06 >20.0
27.5 5.8 2.1 >2.5 5.9 3.6 5.3 >2.4 >2.7
6.6 4.2 4.5 12.2 1.1 24.2 20.7 NA 6.8
40.5 24.78 19.8 >49.4 8.2 >53.1 >57.7 – >20.0
6.2 5.9 4.4 >4.1 7.4 >2.2 >2.8 – >3.0
positive control; CD: concentration that doubles the QR activity; IC50: concentration that inhibits 50% of cell growth; CI: index which correspond to IC50/CD; ND: not determined; NA: not active.
5
to the metabolically more stable methoxy group in position 7 instead of an acetoxy, which is likely to be hydrolysed by lipases. Icetaxane and dehydroabietane quinones provide new scaffolds for QR inducing agents due to their fully substituted quinone moiety. Although fruticulin A (1) and its demethyl derivative (demethyl fruticulin A – 2) are known to induce apoptosis in cancer cell lines (Giannoni et al., 2010; Monticone et al., 2010), these compounds may still be interesting leads to the development of more potent QR inducers.
2.5. HDAC inhibition of compounds from S. corrugata A 100 lg/ml solution of the exudate inhibited 48% of HDAC activity from HeLa nuclear extracts, and the isolated a-hydroxylb-isopropylquinones were further tested. The results showed that 1, 2 and 3 were the most active ones (Table 4). Several natural products, such as isothiocyanates (cruciferous vegetables), butyrate (fiber, butter, and oil), organosulfur compounds (garlic, onion, and broccoli), and organoselenium compounds (seleniferous vegetables) exhibit similar HDAC inhibitory activities (Ho et al., 2012). HDAC inhibition was shown to decrease the proliferation and survival of cancer cells (Glozak and Seto, 2007; Potthoff and Olson, 2007). Thus, according to our findings, HDAC inhibition by demethyl-fruticulin A (2) may be one of the mechanisms behind its antitumor activity previously demonstrated (Monticone et al., 2010). To the best of our knowledge, this is the first report of HDAC inhibition by a-hydroxyquinones, which might be explained by the ability of this particular quinone to bind tightly its zinc active site though the presence of the two close oxygen atoms (Yamada et al., 2004). Because HDAC selectivity is important, the activity of 1–3 was tested against various isoforms. The inhibitory profile of 3, with a selectivity against HDAC6, was different from the ones of 1 and 2. In comparison to the latter compounds, which were active on HDAC1, HDAC3 and to a lower extend on HDAC6, 3 was slightly active on HDAC2 and active on HDAC6. The presence of a methoxy group in the 7-membered ring is the main difference between 3 and 1. These results are interesting, as HDAC6 is a promising target in oncogenic cell transformation (Aldana-Masangkay and Sakamoto, 2011), and finding new specific inhibitors could contribute to a better understanding of the role of HDAC6 in carcinogenesis.
3. Conclusion Little information was available on the phytochemical constituents of S. corrugata, besides its major constituents. NP-HPLC analysis showed that several benzoquinones were present in the exudate. A complementary NMR profile indicated that in addition to icetexanes benzoquinones a second type of abietane, namely a-hydroxy-b-isopropyl-benzoquinones, were present. These results, in addition to in vitro information on QR induction activity, motivated the bioactivity-guided isolation of constituents from the exudate. Hence, four new compounds were isolated and identified: fruticulin C (3), 7a-methoxy-19-acetoxy-royleanone (4), 7a,19-diacetoxy-royleanone (5) and 7-dehydroxy-conacytone (7) in addition to three known a-hydroxy-b-isopropyl-benzoquinones. All these quinones were found to be inducers of QR, amongst which three (1, 2 and 5) showed an activity in the low lM range. The HDAC inhibition was not as potent but an interesting subtype selectivity was observed. All of these results revealed key structural elements to further explore similar compounds for cancer chemoprevention.
Please cite this article in press as: Giacomelli, E., et al. Cancer chemopreventive diterpenes from Salvia corrugata. Phytochemistry (2013), http://dx.doi.org/ 10.1016/j.phytochem.2013.09.011
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Table 4 HDAC inhibition of selected isolated diterpenes. Compounds
1 2 3 4 5 6 7 Trichostatin Ab
HeLa nuclear extract
HDAC1
HDAC2
HDAC3
HDAC6
% inhibition (100 lg/ml)
IC50 [lM]
IC50 [lM]
IC50 [lM]
IC50 [lM]
IC50 [lM]
100.0 100.0 100.0 26.4 36.9 56.8 55.6 ND
72.8 ± 9.6a 76.7 ± 7.3a >200a ND ND ND ND 20.2 nM
59.5 ± 5.7 37.2 ± 3.5 >200 ND ND ND ND 6.5 ± 0.1 nM
>200 >200 122.4 ± 5.9 ND ND ND ND 9.2 ± 1.4 nM
40.9 ± 9.8 40.9 ± 0.3 >200 ND ND ND ND 35.9 ± 5.2 nM
91.1 ± 23.4 79.8 ± 16.3 42.8 ± 5.3 ND ND ND ND 1.5 ± 0.6 nM
ND: not determined. a n = 2. Except otherwise stated n = 3. b Positive control.
4. Experimental 4.1. General procedures High-resolution MS spectra were recorded on a Q-TOF Micromass (Waters). Samples dissolved in chloroform/methanol/water 2:7:1 containing 5 mM ammonium acetate were mixed in 1:3 proportions with leucine-enkephalin (for internal calibration of the MS spectra; Sigma–Aldrich, Steinheim, Germany) in acetonitrile (100 lg/ml). This mixture was infused using a TriVersa NanoMateÒ (Advion) (Zhang et al., 2004). The NanoMate contains microfabricated nanoESI nozzles, which operated at solution flow rates of 83 nL/min. The nanoESI was initiated by applying 1.6 kV to the pipette tip and 0.5 psi (nitrogen) gas pressure on the liquid. The m/z range was set to be 100–1000 Da in centroid mode with a scan time of 0.25 s and an inter-scan delay of 0.01 s. NMR analyses were performed on an Agilent Varian Unity Inova 500 MHz NMR instrument (Palo Alto, CA, USA). All samples were dissolved in deuterated chloroform and chemical shifts were reported in parts per million (d) using the residual chloroform signal as internal standard (dH 7.26, dC 77.2). Coupling constants (J) were reported in Hz. 4.2. Plant material Fresh aerial parts of Salvia corrugata Vahl. were collected in November 2010 by the Centro Regionale di Sperimentazione ed Assistenza Agricola (Albenga, Italy). The species was identified by Dr. Gemma Bramley and a voucher specimen is deposited in Kew Herbarium (K) where it is referred as ‘interreg III alcotra Project N°074’. 4.3. Extraction The extraction was done according to a previously published protocol (Bisio et al., 2008). The fresh aerial parts (135 g) of S. corrugata Vahl. were immersed for 20 s in an appropriate volume of dichloromethane to fully cover the material. After filtration, the extraction solvent was removed under reduced pressure to give 1.5 g of crude exudate material. 4.4. NP-HPLC-PDA-ELSD analysis of crude exudate, fractions, and purified compounds Crude exudate, fractions and pure compounds were analysed by NP-HPLC-PDA-ELSD with an HP-1050 liquid chromatography system (Hewlett Packard) consisting of quaternary pumps, an inline degasser, an auto-sampler, and a photodiode array detector (PDA) connected to a light scattering detector (500 ELSD, Alltech)
and an HP interface (35900, Hewlett Packard). An analytical silica column (Cosmosil 5Si-II, 4.6 250 mm, 5 lm) was used. n-Hexane (HPLC grade, solvent A) and ethyl acetate (HPLC grade, solvent B) were used as eluent at a flow rate of 1.0 ml/min. PDA wavelengths were 270 and 450 nm and the ELSD was set at 60 °C with a SLPM (Standard Liter Per Minute) of 0.75. Usually, samples of 10 ll were injected at a concentration of 10 mg/ml for extracts and fractions and at 1 mg/ml for purified compounds. A gradient composed of an initial isocratic step at 5% B for 5 min followed by an increase to 100% B over 60 min was used to profile the exudate. A 30 min gradient from 0% to 100% B was used to control the fraction content and the compound purity.
4.5. Isolation The exudate (1.5 g) was separated into 9 fractions by High Speed Counter Current Chromatography (HSCCC) consisting of an HSCCC unit (TBE300B; Tauto, modified by Sertec, Switzerland) and two pumps (LC-10AD, Shimadzu). The coil volume was 260 ml. Solvent systems from the Arizona solvent systems were used (Berthod et al., 2005), with the upper phase as stationary phase and the lower phase as mobile phase in the head-to-tail elution mode, at a flow rate of 7 ml/min. The column was first filled with the upper phase of the biphasic mixture, then the coil was rotated in a reverse direction at a constant speed of 720 rpm, and the system equilibrated with lower mobile phase (aqueous–methanol phase) up to the head of the coil in head-to-tail mode. The equilibration point of the system was determined when no more stationary phase was eluted (hydrodynamic equilibration). Fractions of 100 mL were collected. After the final gradient step, rotor rotation was stopped and the column content (organic and aqueous phases) was ‘‘washed-off’’. The exudate was fractionated using the solvent system P (heptane/ethyl acetate/methanol/water 6:5:6:5 v/v) of the Arizona solvent systems for HSCCC (Berthod et al., 2005). 1.5 g were dissolved in 7 mL of lower phase (heptane/ethyl acetate/methanol/water 0.11:5:19.2:36.9 v/v) and 7 mL of upper phase (heptane/ethyl acetate/methanol/water 69.4:22.8:43.8:0.4 v/v). 9 fractions were obtained (P1 to P9). The QR induction ratio of each fraction was 1.9 (5 lg/ml), 7.2 (5 lg/ml), 6.6 (5 lg/ml), 6.2 (5 lg/ml), 5.8 (5 lg/ ml), 9.8 (5 lg/ml), 8.9 (10 lg/ml) and 11.4 (5 lg/ml) for fractions P2 to P9, respectively. Compounds 1–7 were purified by repeated preparative NPHPLC with a semi-preparative NP-HPLC apparatus composed of a binary pump (System Gold High Performance Liquid Chromatograph P127, Beckman) and a UV–Visible detector (HP1050, Hewlet Packard) using a semi-preparative silica column (Cosmosil 5Si-II, 10 250 mm, 5 lm) with a pre-column (Cosmosil 5Si-II, 10 20 mm, 5 lm). Elution was achieved at a flow rate of
Please cite this article in press as: Giacomelli, E., et al. Cancer chemopreventive diterpenes from Salvia corrugata. Phytochemistry (2013), http://dx.doi.org/ 10.1016/j.phytochem.2013.09.011
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4.7 ml/min using n-hexane (HPLC grade, solvent A) and ethyl acetate (HPLC grade, solvent B). The LC protocols were optimised at the analytical scale and geometrically scaled up to give the semipreparative LC procedures (Guillarme et al., 2008, 2009). Fractions P4 (454.2 mg), P7 (24.5 mg), and P9 (115.2 mg) were further purified by semi-preparative NP-HPLC. Compound 1 (15.9 mg, tR: 9.0 min) was purified from fraction P4 (an isocratic step at 22% B for 7.4 min followed by a linear gradient to 100% B in 23.4 min). Compound 6 (1.6 mg, tR: 11.0 min) was purified from fraction P7 (an initial linear gradient from 15% B to 22% B in 5.3 min, followed by a second gradient steps to 50% B in 20.5 min). Compounds 2 (16.9 mg, tR: 8.2 min), 4 (1.2 mg, tR: 9.0 min), 7 (1.2 mg, tR: 11.0 min), 5 (3.9 mg, tR: 11.5 min) and 3 (1.4 mg, tR: 12.1 min) were purified from fraction P9 (an isocratic step at 11% B for 4.1 min followed by a linear gradient to 100% B in 26.7 min). The purity of each purified compound was assessed using NP-HPLCELSD and NMR, and only compounds with no impurities observed were used for further biological evaluation. When necessary, additional fractionation steps were used to fully purify the compounds of interest. 4.5.1. Fruticulin C (3) Compound 3 is a red powder with [a]25D 54.7° (c 0.7, CHCl3). This indicated that 3 did not occur as a racemic mixture. HR-()MS: m/z = 353.1382 [MH], calculated for C21H22O5: 354.1467 Da; IR: 3370, 1706, 1639, 1605, 1466, 772; UV/Vis kmax(CH3OH) nm (log e): 238 (4.99); 1H (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 1 and 2. 4.5.2. 7a-Methoxy-19-acetoxy-royleanone (4) Compound 4 is a yellow powder with [a]25D 60.9° (c 0.7, CHCl3); HR-()-MS: m/z = 403.2168 [MH], calculated for C23H32O6: 404.2199 Da; IR: 3347, 1733, 1650, 1637, 1607, 1458, 1373, 1245, 983, 755; UV/Vis kmax(CH3OH) nm (log e): 270 (4.40); 1 H (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 1 and 2. 4.5.3. 7a,19-Diacetoxy-royleanone (5) Compound 5 is a yellow/orange powder with [a]25D -37.7 (c 1.00, CHCl3); HR-()-MS: m/z = 431.1939 [MH], calculated for C24H32O7: 432.2148 Da; IR: 3357, 1739, 1660, 1636, 1609, 1458, 1373, 1234, 984, 757; UV/Vis kmax(CH3OH) nm (log e): 257 (3.46); 1 H (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Tables 1 and 2. 4.5.4. 7-dehydroxy-conacytone (7) Compound 7 is a yellow powder with [a]25D 53.2 (c 0.4, CHCl3); HR-()-MS: m/z 345.1661 [MH], calculated for C20H26O5: 346.1780 Da; IR: 3140, 1775, 1745, 1735, 1500, 880; UV/Vis kmax(CH3OH) nm (log e): 276 (3.95); 1H (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3), see Tables 1 and 2.
7
PowerWave HT microplate spectrophotometer (BioTek Instruments, Luzern, Switzerland) at 595 nm. In parallel, the amount of viable cells was determined by protein quantification using crystal violet staining and measurement of the absorption at 595 nm on the aforementioned spectrophotometer. Extracts and fractions were tested at a fixed concentration of 20 lg/ml in single three-well experiments whilst dose–response curves were the mean of independent triplicates. Isoliquiritigenin and 4’-bromoflavone were used as positive controls. The induction ratios were measured, and CD values (concentration corresponding to an induction ratio of 2), as well as the cytotoxicity (expressed as IC50) were calculated using GraphPad Prism 5 software. Finally, the chemopreventive index (CI) was calculated by dividing the IC50 by the corresponding CD value. 4.7. Histone deacetylase inhibition (HDAC) The crude exudate and the isolated constituents were tested for HDAC inhibition on nuclear cell extracts from HeLa cells, as well as HDAC specific isoforms 1–3 and 6 (all from Enzo Life Sciences, Lausen, Switzerland). Reactions were carried out in 96-well half microtiter plates. The HeLa nuclear extract or specific isoforms (HDAC1-3 and 6) were diluted in assay buffer (50 mM Tris at pH 8.0 adjusted with HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2). Test compounds diluted in DMSO were added at a final concentration of 100 lg/ml (5% DMSO in each well) for screening purposes. According to the manufacturer protocol, the reaction was initiated by the addition of the Fluor de Lys™ specific substrate (Enzo Life Sciences, Plymouth Meeting, PA, USA) followed by incubation at 37 °C for 15 min. The reaction was stopped with 1 lM Trichostatin A. Fluor de Lys developer™ I or II was added 10 min prior to fluorimetric detection. HDAC inhibition was calculated by comparing the amount of deacetylated substrate between control (100% HDAC activity) and test sample. The relative amounts of deacetylated substrate were obtained by fluorescence reading with excitation at 360 nm and emission at 460 nm for HeLa nuclear extract and HDAC1, 3, 6 isoforms. Excitation at 485 nm and emission at 528 nm was used for HDAC2. Dose response curves were obtained to determine the IC50 of active compounds. Measurements were done in triplicate. Trichostatin A was used as positive control. Acknowledgement The authors thank Olivier Ciclet for his help in the recording of the HR-MS spectra. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 09.011.
4.6. Quinone reductase induction activity The Hepa1c1c7 cell line and its clone c35 (ATCC, Rockville, MD, USA) were cultured according to the ATCC recommendations in a-modified Minimal Essential Medium (Life Technologies, Zug, Switzerland) containing 2 mM glutamine, supplemented with 100 units/ml penicillin, 100 lg/ml streptomycin, and 10% foetal calf serum at 37 °C, 5% CO2, and humidified atmosphere. The method described by Kang and Pezzuto (2004) was used. In brief, quinone reductase activity was determined in a 96-well format after two days sample incubation. Cells were then lysed and the NADPH-dependent menadiol-mediated reduction of 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to the corresponding blue fromazan was measured on a
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