Phytochemistry xxx (2014) xxx–xxx
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Antibacterial compounds from Salvia adenophora Fernald (Lamiaceae) Angela Bisio a,⇑, Anna Maria Schito b, Samad Nejad Ebrahimi c,d, Matthias Hamburger c, Giacomo Mele a, Gabriella Piatti b, Giovanni Romussi a, Fabrizio Dal Piaz e, Nunziatina De Tommasi e a
Dipartimento di Farmacia, Università di Genova, Via Brigata Salerno 13, 16147 Genova, Italy Dipartimento di Scienze Chirurgiche e Diagnostiche Integrate, Sezione di Microbiologia, Università di Genova, Largo Rosanna Benzi 8, 16145 Genova, Italy c Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland d Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran e Dipartimento di Farmacia, Università di Salerno, Via Giovanni Paolo II 132, 84084 Salerno, Italy b
a r t i c l e
i n f o
Article history: Received 8 June 2014 Received in revised form 3 October 2014 Available online xxxx Keywords: Lamiaceae Salvia adenophora Fernald ECD Clerodane diterpenes Derivatives of 12-oxo-phytodienoic acid Antimicrobial activity
a b s t r a c t From the aerial parts of Salvia adenophora Fernald four derivatives of 12-oxo-phytodienoic acid (1–4) together with five clerodane diterpenoids (5, 6, 8–10), and one known diterpene (7) have been isolated. Compounds 1–6 and 8–10 are described for the first time. The structures were established by extensive 1D, 2D NMR and HRESI-TOFMS spectroscopic methods. Finally, the absolute configuration has been established by comparing of experimental and quantum chemical calculation of ECD spectra. Despite a total lack of antimicrobial activity of the plant extract, hinting to the existence of antagonistic interactions in the crude material, three oxylipins (2–4) displayed a promising inhibition on Gram-positive multidrug-resistant clinical strains including Staphylococcus aureus, Streptococcus agalactiae and, particularly, Staphylococcus epidermidis, while the compounds 9 and 10 revealed a specific and strain-dependent activity against S. epidermidis. Interestingly, the inhibition provided by these compounds was independent of the resistance patterns of these pathogens to classic antibiotics. No action was reported on Gram-negative strains nor on Candida albicans. These results confirm that clerodanes and, particularly, prostaglandin-like compounds can be considered as interesting antimicrobial agents deserving further study. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The worldwide dramatic increase in nosocomial and/or community acquired infections caused by resistant or multi-resistant Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-resistant Enterococci (VRE) (mainly Enterococcus faecium and Enterococcus faecalis) is one of the most serious and urgent public-health challenge of the new millennium (Calfee, 2012; Lentino et al., 2008; Otto, 2009). These microorganisms are considered commensal bacteria that constitute a major component of the normal microflora of humans. In addition to their commensal role however, these species also behave frequently as nosocomial pathogens in immune compromised and critically ill patients, where they cause infections ranging from minor ailments to severe hospital infections and life-threatening conditions like pneumonia, meningitis and bacteriemia (Calfee, 2012; Otto, 2009). Besides being naturally
⇑ Corresponding author. Tel.: +39 010 3532637; fax: +39 010 3532684. E-mail address:
[email protected] (A. Bisio).
refractory to antibacterial drugs, such as the Enterococci, many of these species are well known for their capability to rapidly develop acquired resistance (Fisher and Phillips, 2009). They may not only loose susceptibility to single drugs but, quite often, may also acquire resistance to multiple drugs. Moreover, resistance traits may spread very quickly both in community and in hospital settings, requiring additional costs for the diagnosis and therapy of the serious infections that these pathogens are capable to sustain (Eurosurveillance, 2012; French, 2010). Hence, there is an urgent need for new classes of antibacterial compounds, with activity against these resistant or multi-resistant strains. Natural substances produced by plants that are traditionally used for the treatment of bacterial infections are of interest as potential new antibacterials. The genus Salvia, in particular, has proven to be a good source for antibacterial compounds (Gibbons, 2004; Itokawa et al., 2008; Kamatou et al., 2008). We recently described the antibacterial and anti-biofilm activity of two diterpene quinones from the exudation product of the fresh aerial parts of Salvia corrugata Vahl. against several Gram positive strains (Bisio et al., 2008; Schito et al., 2011). Prompted by these positive results we investigated Salvia adenophora Fernald, a Mexican species belonging to the
http://dx.doi.org/10.1016/j.phytochem.2014.10.033 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bisio, A., et al. Antibacterial compounds from Salvia adenophora Fernald (Lamiaceae). Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.033
2
A. Bisio et al. / Phytochemistry xxx (2014) xxx–xxx
subgenus Calosphace, section Nobiles. The species is cited by Hunn in the Zapotec Natural History (Hunn, 2014), within a group of other species, not all belonging to the same genus, with red, orange, yellow, or even blue flowers, attractive to hummingbirds, used for child’s rash or to ‘‘clean’’ the body. Herein we report the phytochemical investigation of the exudation mixture of the fresh aerial parts of S. adenophora, and the evaluation of antimicrobial activity against multidrug-resistant human Gram-positive pathogens of the isolated compounds. 2. Results and discussion 2.1. Chemical investigation of the secreted material The dichloromethane soluble portion of the exudate mixture of S. adenophora was separated by silica gel column chromatography and RP-HPLC to afford nine compounds (1–6, 8–10) along with one known clerodane diterpene, 2-hydroxyhardwickiic acid (7) (Jolad et al., 1988) (Fig. 1) and a mixture of ursolic and oleanolic acids (Seebacher et al., 2003) identified by their physical and spectroscopic data, which were largely consistent with those published in the literature. The molecular formulas of C19H30O4 ([M+H]+ 323.2229) for compounds 2 and 3 and C21H32O5 ([M+H]+ 365.2343) for compounds 1 and 4 were determined by HRESI-TOFMS. The IR spectrum of compounds 1–4 showed bands at 3007–3006, 2930– 2927, 2857–2855, 1709–1706, 1588–1587 that were characteristic of methyl 12-oxophytodienoate (Chu et al., 1995), along with band indicative of free OH groups (3474 and 3449 cm1) in compounds 2 and 3. The neutral loss of methyl formate observed in the ESI-MS/ MS spectra of 1–4 revealed the presence of a methyl ester group. Moreover, a fragment ion at m/z 207 that was generated by the
elimination of acrolein from the ion at m/z 263 suggested the presence of an a,b-unsaturated ketone function. Ketene elimination in the spectra of compounds 1 and 4 was indicative of an acetyl moiety esterified to a hydroxyl-group. The 1D and 2D-NMR spectral data (Table 1) allowed the assignment of all the hydrogen and carbon signals, including 4 olefinic protons, 8 CH2, 2 CH3, 3 CH and 2 C groups for compounds 2 and 3, and 4 olefinic protons, 8 CH2, 3 CH3, 3 CH and 3 C groups for compounds 1 and 4. The presence of a conjugated ketone was corroborated by the low field signals at dH = 7.60 (m, 1H) and 6.12 (d, J = 5.6 Hz, 1H) for compounds 1 and 2, and at dH = 7.73 (dd, J = 5.5, 2.4 Hz, in 3 and m in 4, respectively, 1H) and 6.18 (d, J = 5.5 Hz, in 3 and m in 4 respectively, 1H) for compounds 3 and 4. These 1H signals and the 13C resonances (dC = 211.9, 167.6, 133.0, 51.6, 47.2 for compounds 1 and 2; 210.9, 167.1, 132.6, 50.0, 44.4 for compounds 3 and 4), as well as HSQC, HMBC and 1H–1H COSY signals indicated a five membered ring for all these compounds. Inspection of COSY, TOCSY, HSQC, HMBC spectral data revealed two additional structural fragments. The first fragment was established as CH3AOACOACHACH2ACH2ACH2ACH2ACH2A [dC = 171.0 (C1), 72.4 (C2), 31.2 (C3), 25.2 (C4), 29.1 (C5), 29.6 (C6), 27.5–27.7 (C7) for compounds 1 and 4, and dC = 176.0 (C1), 70.5 (C2), 34.1 (C3), 24.9 (C4), 29.3 (C5), 29.7 (C6), 27.5–27.7 (C7) for compounds 2 and 3], and the second fragment as ACH2ACH@CHACH2ACH3 [dC = 28.4 (C14), 125.2 (C15), 134.1 (C16), 20.7 (C17), 14.4 (C18) for compounds 1 and 2 and dC = 24.0 (C14), 127.1 (C15), 134.1 (C16), 20.7 (C17), 14.4 (C18) for compounds 3 and 4]. Diagnostic HMBC correlations H8AC6, H8AC7, H8AC10, H9AC8, H10AC8, H13AC8 for compounds 1 and 2, and H6AC7, H7AC8, H8AC6, H8AC7, H8AC10, H13AC8 for compounds 3 and 4 allowed to link the first substructure at C9 of the five membered ring. On the other hand, HMBC correlations H9AC14, H11AC13, H14AC12, H13AC14, H13AC15,
Fig. 1. Structural formulas of compounds isolated from Salvia adenophora.
Please cite this article in press as: Bisio, A., et al. Antibacterial compounds from Salvia adenophora Fernald (Lamiaceae). Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.033
13
s
m m, 2.49 m m m m, 2.06 m m s
– 4.99 1.81 1.37 1.31 1.25 1.36 1.15 2.98 7.73 6.18 – 2.44 2.13 5.36 5.44 2.05 0.96 3.74 – 2.14 171.0 72.4 31.2 25.2 29.1 29.6 27.7 30.9 44.4 167.1 132.6 210.9 50.0 24.0 127.1 134.1 20.7 14.4 52.4 170.7 20.8 m m, 2.49 m m m m, 2.06 m t (7.3, 7.3) s
– 4.19 1.64 1.38 1.33 1.25 1.36 1.15 2.98 7.73 6.18 – 2.44 2.13 5.36 5.44 2.05 0.96 3.79 – – 176.0 70.5 34.1 24.9 29.3 29.7 27.7 30.9 44.4 167.1 132.6 210.9 50.0 24.0 127.1 134.1 20.7 14.4 52.7 – – m m, 2.46 m m m m, 2.06 m t (7.3, 7.3) s s
HMBC correlations are from proton(s) stated to the indicate carbon(s). a
m m, 2.46 m q (7.9, 7.9, 7.6) q (7.5, 7.5, 7.4) m, 2.06 m t (7.5, 7.5) s
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
171.0 72.4 31.2 25.2 29.1 29.6 27.5 34.5 47.2 167.6 133.0 211.9 51.6 28.4 125.2 134.1 20.7 14.4 52.4 170.7 20.8
– 4.99 1.81 1.37 1.31 1.25 1.32 1.49 2.58 7.60 6.12 – 2.01 2.28 5.26 5.45 2.05 0.96 3.74 – 2.14
m m, 1.82 m m, 1.38 m m, 1.33 m m, 1.34 m m, 1.36 m m, 1.50 m m m nd (5.6)
– 1, 3, 4, 20 1, 2, 4, 5 2, 3, 5, 6 3, 4, 6 5, 7 5, 6, 8 6, 7, 9, 10, 13 8, 10, 11, 13, 14 8, 9, 11, 13 9, 10, 12, 13 – 8, 10, 12, 14, 15 9, 12, 13, 15, 16 13, 14, 17 14, 17, 18 15, 16, 18 16, 17 1 – 2, 20
176.0 70.5 34.1 24.9 29.3 29.7 27.5 34.5 47.2 167.6 133.0 211.9 51.6 28.4 125.2 134.1 20.7 14.4 52.7 – –
– 4.19 1.64 1.38 1.33 1.25 1.32 1.49 2.58 7.60 6.12 – 2.01 2.28 5.26 5.45 2.05 0.96 3.79 – –
m m, 1.78 m, 1.44 m, 1.35 m, 1.34 m, 1.36 m, 1.50 m m d (5.6)
m m m m m m
– 1, 3, 4 1, 2, 4, 5 3, 5, 6 4, 6 5, 7 5, 6 6, 7, 9, 10 8, 10, 14 9, 11, 13 9, 10, 12, 13 – 8, 9, 12, 14, 15 9, 12, 13, 15, 16 14, 17 17, 18 15, 16, 18 16, 17 1 – –
dC HMBCa dH dC
2
HMBCa dH dC
1 Position
Table 1 C NMR (125 MHz) and 1H NMR (600 MHz) of compounds 1–4 in CDCl3 (d in ppm, J in Hz) (n = nearly).
3
dH
m m, 1.78 m m, 1.44 m m, 1.35 m m, 1.34 m m, 1.39 m m, 1.75 m m dd (5.5, 2.4) d (5.5)
– 1, 3, 4 1, 2, 4, 5 5, 6 4, 6, 7 5, 7, 8 5, 6, 8 6, 7, 10 – 9, 11, 12, 13 9, 10, 12, 13 – 8, 9, 12, 14, 15 9, 12, 13, 15, 16 13 17, 18 15, 16, 18 16 1 – –
dC HMBCa
4
dH
m m, m, m, m, m, m, m m m
1.82 1.38 1.33 1.34 1.39 1.75
m m m m m m
– 1, 3, 4, 20 1, 2, 4, 5 2, 3, 5, 6 3, 4, 6 5, 7, 8 5, 6, 8 6, 7, 9, 10 – 8, 9, 11, 12, 13 9, 10, 12, 13 – 8, 9, 10, 12, 14, 15 9, 12, 13, 15, 16 13, 14, 17 14, 17, 18 15, 16, 18 16, 17 1 – 2, 20
HMBCa
A. Bisio et al. / Phytochemistry xxx (2014) xxx–xxx
3
H14AC13 for compounds 1 and 2, and H11AC13, H13AC12, H13AC14, H13AC15, H14AC12, H14AC13, H15AC13 for compounds 3 and 4 allowed to link the second fragment to C13 of the five membered ring. For 1–4, a Z configured double bond at C15AC16 was established on the basis of chemical shifts (Table 1), ROESY correlations H14AH17, and comparison with literature data (Baertschi et al., 1988; Chu et al., 1995). Comparison of NMR spectra of compounds 1 and 4 with those of compounds 2 and 3 indicated the presence of an acetyl moiety esterified to the HOAC2 group (Table 1). These data were consistent with those of derivatives of oxophytodienoic acid (Bohlmann et al., 1982, 1983). The data of H9 and H13 were largely consistent with those published for 12-oxo-cis-10,15-phytodienoic acid and its methyl ester (Ainai et al., 2003; Baertschi et al., 1988; Crombie and Mistry, 1991; Kobayashi and Matsuumi, 2002) (compounds 3 and 4), and for 12-oxo-trans-10,15-phytodienoic acid and its methyl ester (compounds 1 and 2), respectively (Ainai et al., 2003; Baertschi et al., 1988; Bohlmann et al., 1982; Chu et al., 1995; Kobayashi and Matsuumi, 2002). Moreover, the signals of H10 and H11, i.e. dH = 7.73 and 6.18 for compounds 3 and 4, and dH = 7.60 and 6.12 for 1 and 2 were consistent with literature data for 12-oxo-cis-phytodienoic acid and its stereoisomer (Ainai et al., 2003; Kobayashi and Matsuumi, 2002). The relative and absolute configurations at C9 and C13 of 1–4 were established in a next step. The 13C NMR chemical shift of C9 and C13 in 1 and 2 were 47.2 and 51.6 ppm, and 44.4 and 50.0 ppm in 3 and 4 (Table 1). Thus, given that the compounds had been isolated by non-enantioselective separation methods, they represented two pairs of diastereomers (1 and 3, and 2 and 4). Electronic circular dichroism (ECD) spectra were calculated for possible stereoisomers of 1–4, and compared with experimental spectra (Figs. 2 and 3). In the experimental ECD spectra of 1–4 a positive Cotton effect (CE) was observed at 225 nm (Figs. 2 and 3) which was due to the p ? p⁄ transition of the cyclopentenone ring. The calculated ECD spectra of 1–4 showed a positive CE at 225 nm for the 9S configuration. The simulated spectrum of the (9S, 13R) stereoisomer showed negative and positive CEs at 205 and 220 nm, respectively, and fitted well with theexperimental spectrum of 1 (Fig. 2). The ECD spectra of 3 and 4 were virtually identical and showed a better match with the spectrum calculated for the (9S, 13S) stereoisomer (Fig. 3). The experimental ECD spectrum of 2 was slightly different from that of 1, as it lacked of a negative CE at 205 nm. The difference between calculated and experimental spectra presumably resulted from minor differences between calculated and solution conformers of this highly flexible molecule (Fig. 4). The configuration of the cyclopentenone ring in
Fig. 2. Experimental ECD spectra of 1 and 2, and calculated spectra of the 9S,13R stereoisomer.
Please cite this article in press as: Bisio, A., et al. Antibacterial compounds from Salvia adenophora Fernald (Lamiaceae). Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.033
4
A. Bisio et al. / Phytochemistry xxx (2014) xxx–xxx
Fig. 3. Experimental ECD spectra of 3 and 4, and calculated spectra of the 9S,13S stereoisomer.
oxylipins has been previously studied by stereoselective synthesis of 12-oxo-cyclopentenoic acid derivatives and comparison of their optical rotation. The [a]D values of (9S, 13S) stereoisomers were smaller than those of (9S, 13R) stereoisomers (Kobayashi and Matsuumi, 2002). Specific optical rotations for compounds 1–4 were +35.5, +29.9, +60.0, and +107.3, respectively. Taking into account the sign and the magnitude of optical rotation together with the ECD and NMR data, the absolute configuration of the cyclopentenone ring was established as (9S, 13R) in oxylipins 1 and 2, and as (9S, 13S) in 3 and 4. Thus compounds 1–4 were identified as: methyl (9S,13R)-2-acetoxy-12-oxo-(10Z,15Z)-phytodien oate (1), methyl (9S,13R)-2-hydroxy-12-oxo-(10Z,15Z)-phytodienoate (2), methyl (9S,13S)-2-hydroxy-12-oxo-(10Z,15Z)-phytodienoate (3), methyl (9S,13S)-2-acetoxy-12-oxo-(10Z,15Z)-phytodi enoate (4). Oxylipins are a diverse group of lipid-derived signaling compounds, found in all organisms (Jahn et al., 2008), involved in plant growth and development (Tian et al., 2012). Plant oxylipins have been described as free oxylipins (Bohlmann et al., 1982, 1983; Ohashi et al., 2005; Tamayo-Castillo et al., 1989; Zdero et al., 1991), as oxylipins esterified with a number of other molecules in the cell, like glycerolipids (glycolipids, phospholipids,
and neutral lipids) (Andersson et al., 2006; Bottcher and Weiler, 2007; Buseman et al., 2006; Göbel and Feussner, 2009; Stelmach et al., 2001), and oxylipins conjugated to amino acids and other metabolites, such as sulfate, glutathione, ethanolamine, and carbohydrates (Mosblech et al., 2009). The compounds present in the exudate material of S. adenophora were free oxylipins. Compound 5 was isolated as an amorphous powder. Its molecular formula was determined to be C22H30O5 by HRESI-TOFMS (negative mode) (m/z 373.4598 [MH], calcd. for: 373.4626). The 1D and 2D-NMR data (Table 2) were consistent with the data reported for 2-acetoxyhardwickiic acid (Singh et al., 1998) confirming for 5 the same planar structure. The slight differences in the NMR spectra and the specific optical rotation (5: [a]25 D : 52.0, c 1.00, CHCl3; 2-a-acetoxyhardwickiic acid: 1.1, c 2.07, CHCl3, literature) led us to hypothesize a different configuration at C2. Compound 6 (HRESI-TOFMS m/z 359.2217 [MH]) had a molecular formula of C22H32O4, equating to seven double bond equivalents. The UV spectrum showed an absorption band at 208 nm, and the IR spectrum (KBr) exhibited stretching frequencies for OH (3434 cm1), C@O (1736 cm1), and CH@CH (1638 cm1) functionalities. The 13C NMR spectrum showed resonances for four methyl, six methylene, seven methine, and five quaternary carbons including a carboxyl group which were indicative of a clerodane diterpene skeleton (Table 2) (Fujita, 1971; Merrit and Ley, 1992). The 13C NMR signals at dC = 170.9 (C18), 135.9 (C3) and 144.2 (C4), the 1H NMR data [dH = 6.76 (H3), and HMBC correlations H3AC4 and H3AC18 were indicative of an a,b-unsaturated carboxyl group. The 1D TOCSY and COSY spectra suggested a C3AC10 spin system and the presence of an ethoxy group at C2 (Table 2) that was confirmed by HMBC correlations H2AC10 , H10 AC2, H10 AC20 , H20 AC10 . The six-carbon side chain (C11 through C16) of the clerodane skeleton contained a terminal b-substituted furan ring, as was corroborated by IR (absorptions bands at 1504 and 870 cm1) and NMR data (Table 2). Diagnostic ROESY correlations H8AH10, H17AH19, H17AH20 and H19AH20 indicated their spatial proximity. On the basis of the 13C NMR chemical shift of the angular methyl (C19; dC = 19.0), the A/B ring junction was deduced to be trans. In cis clerodanes, this carbon appears downfield by approx. 10 ppm (Choudhary et al., 2010; Manabe and Nishino, 1986; Puebla et al., 2005; Wang et al.,
Fig. 4. Superimposed conformers of compound 1 obtained from conformational analysis using OPLS-2005 model, the compound shows high flexibility.
Please cite this article in press as: Bisio, A., et al. Antibacterial compounds from Salvia adenophora Fernald (Lamiaceae). Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.033
Position
5 dC
HMBCa
8
dC
dH
HMBCa
1.93 n d (14.11), 1.56 m 3.89 n t (3.9)
2, 3, 5, 9, 10
9 dH
HMBCa
27.5
1.73 m, 1.88 m
2, 3, 9, 10
1, 3, 4, 10, 10
64.5
4.40 m
3, 4, 10
dC
10 dH
HMBCa
17.6
1.47 m ax, 1.67 m eq
2, 3, 4, 5, 9, 10
27.4
2.22 m, 2.32 m 6.82 ns
1, 3, 4, 10 2, 18
dC
1
24.8
1.81 m, 1.87 m
2, 3, 9, 10
23.8
2
67.2
5.39 m
5, 10,
71.8
3
132.7
5, 18,
135.9
6.76 d (4.0)
1, 2, 4, 5, 18
136.7
6.73 m
1, 2, 4, 5, 18
140.1
4 5 6
146.5 38.1 35.4
7, 8,
144.2 38.4 35.5
– – 5, 7, 10, 19
144.0 38.3 35.5
– – 5, 7, 8, 10, 19
141.3 38.9 35.9
7
27.2
8
36.3
6.62 d (3.8) – – 1.23 m, 2.32 m 1.47 m, 1.49 m 1.60 m
1, 3, 4, 10 1, 2, 4, 19 – – 1, 4, 5, 10, 19 5, 6, 8,
9 10
38.5 42.1
– 1.67 m
11
38.6
12
17.7
13 14
125.6 111.0
1.57 1.58 2.34 2.39 – 6.22
15
143.0
16 17
138.4 15.9
18 19 20 10 0
2 a
6 dH
7, 9, 17, 20
36.2
– – 1.23 m, 2.32 m 1.47 m, 1.49 m 1.59 m
– 1, 2, 4, 5, 6, 8, 9, 19, 20 8, 9, 10, 12, 13, 20 9, 11, 13, 14, 16 – 12, 13, 15, 16
38.6 42
– 1.66 n d (13.0)
38.8
126 111.1
1.57 m, 1.58 m 2.30 m, 2.48 m – 6.26 n s
7.34 n s
13, 16
142.8
7.34 n s
6, 7, 9, 10, 11, 17, 20 – 1, 2, 4, 5, 9, 19, 20 9, 10, 12, 13, 20 9, 11, 13, 14, 16 – 12, 13, 15, 16 13, 16
13, 14, 15 7, 8, 9
138.4 16
7.19 n s 0.84 d (6.6)
13, 14, 15 7, 8, 9
172.4 15.8
– 0.83 d (5.6)
– 7, 8, 9
170.9 19.0 18.2
7.18 n s 0.85 d (6.5) – 1.25 s 0.76 s
170.9 19 18.3
– 1.22 s 0.77 s
– 4, 5, 6, 10 8, 9, 10
170.8 18.9 18.2
– 1.22 s 0.76 s
– 4, 5, 6, 10 8, 9, 10, 11
170.4
–
– 4, 5, 6, 10 8, 9, 10, 11, 12 –
3.52 m, 3.62 m 1.18 t (6.9)
2, 20
57.1
3.57 s –
21.3
m, m m, m ns
1.99 s
0
1
9, 17
27.4
17.6
64.8 15.7
6, 9, 17
27.3 36.2
– – 1.23 m, 2.32 m 1.47 m, 1.49 m 1.59 m
38.6 41.4
– 1.71 m
36.4
139.4 142.1
1.42 m, 1.60 m 2.17 m, 2.43 nt (13.1) – 6.79 d (4.4)
102.9
5.73 d (3.4)
13, 14, 16
58.8 61.3 16.1
0
1
18.7
–
5, 6, 8, 9, 17
27.6
7, 9, 11, 20
35.9
– – 1.15 m, 2.40 m 1.44 m, 1.49 m 1.52 m ax
– 1, 2, 4, 5, 6, 8, 9, 11, 19, 20 8, 9, 10, 12, 13, 20 11, 13, 14, 16
39 46.8
– 1.34 m ax
36.4
1.43 m, 1.54 m 1.89 m, 2.02 m – 5.61 t (7.0)
dH
HMBCa
27.6
1.72 m, 1.93 m
2, 3, 5, 9, 10
64.5
4.40 n s
3, 4, 10
136.7
6.72 d (3.81)
– – 4, 7, 8, 19
144.2 38.4 35.6
– – 1.23 m, 2.33 n d (13.2)
5, 6, 8, 9
27.3
1.44 m, 1.50 m
1, 2, 4, 5, 18, 19 – – 4, 5, 7, 8, 10, 19 5, 8, 17
6, 7, 9, 11
36.4
1.51 m
7, 9, 17, 20
– 1, 2, 4, 5, 6, 8, 9, 11, 19, 20 8, 9, 10, 12, 13, 20 11, 13, 14, 16
38.5 41.4
– 1.73 m
36.5
1.42 m, 1.61 n t (12.6)
18.9
2.17 ddd (4.6, 13.5, 13.5), 2.43 n t (13.2) – 7.13 n s
dC
4.21 n d (6.9) 4.18 s 0.81 d (6.5)
13, 14 12, 13, 14 7, 8, 9
175.6 15.9
– 0.84 d (6.0)
– 1, 2, 4, 5, 6, 9, 19 8, 9, 10, 12, 13, 20 11, 13, 14, 16 – 12, 13, 15, 16 12, 13, 14, 16 – 7, 8, 9
170.6 20.7 18.5
– 1.25 s 0.75 s
– 4, 5, 6, 10 8, 9, 10, 11
171.3 18.8 18.2
– 1.22 s 0.76 s
– 4, 5, 6, 10 8, 9, 10, 11
15
–
–
–
–
–
–
–
–
–
–
–
–
–
– 12, 13, 15, 16
29.2 144.8 126.3
– 12, 15, 16
135 144.6 70.6
4.78 n s
A. Bisio et al. / Phytochemistry xxx (2014) xxx–xxx
HMBC correlations are from proton(s) stated to the indicate carbon(s).
5
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Table 2 C NMR (125 MHz) and 1H NMR (600 MHz) of compounds 5, 6, and 8–10 in CDCl3 (d in ppm, J in Hz) (n = nearly).
13
6
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2009). Moreover, the chemical shift of C20 (dC = 18.3) confirmed a cis configuration of methyl groups C17 and C20 (dos Santos et al., 2007). However, the relative configuration at C2 could not be established by NMR experiments, and the relative configuration of 6 was assigned as 5R, 8R, 9S, 10R. The presence of an a,b-unsaturated carboxylic acid next to the stereogenic center at C2 suggested that the absolute configuration of 6 could possibly be solved by electronic circular dichroism (ECD) (Schramm et al., 2013), and likewise also for 5 and 7, which differed only in the substitution of the hydroxy group. The experimental spectrum and calculated ECD spectra of two possible stereoisomers of 6 (2S and 2R) are shown in Fig. 5. The calculated ECD for the 2S stereoisomer showed excellent fit with the experimental data, with two negative CEs at 220 and 245 nm due to p ? p⁄ transition of the a,b-unsaturated carboxylic acid. The calculated spectrum of the 2R stereoisomer (Fig. 5) was distinctly different from the experimental data. Conformational analysis using relative free energies indicated the presence of six stable conformers for the 2S stereoisomer (Fig. 5), differing mainly in the orientation of the side chain and the furan ring. Hence, configuration of 6 was confirmed as 2S, 5R, 8R, 9S, 10R and 6 was identified as (2S,5R,8R,9S,10R)-2-b-ethoxyhar dwickiic acid. The ECD spectra of compound 5 and 7 were identical with that of 6, and we thus concluded that they had the same configuration (Fig. 5). Thus 5 was (2S,5R,8R,9S,10R)-2-b-acetoxyhardwickiic acid. This compound is reported herein for the first time, as previously had been reported only as the 2-a epimer in Grangea maderaspatana (Asteraceae) (Singh and Jain, 1990; Singh et al., 1998). (2S,5R,8R,9S,10R)-2-b-Hydroxyhardwickiic acid (7) has been reported for other genera of Asteraceae (Diplostephium, Laennecia and Conyza) (Jolad et al., 1988; Simirgiotis et al., 2000; Zdero et al., 1992). The relative stereochemistry of these compounds has been reported as a-acetoxy and b-hydroxy, respectively (Misra et al., 1979), and we here established their absolute configuration by ECD spectroscopy. To the best of our knowledge this is the first report on establishing the configuration at C2 in clerodane diterpenoids with the aid of ECD. All compounds showed S-configuration. Compound 8 had a molecular formula of C21H30O6 (HRESI-TOFMS m/z 377.3000 [MH]) implying seven degrees of unsaturation. The 1H NMR spectrum (Table 2) was that of a typical clerodane derivative, with singlets of two tertiary methyls, and a doublet of
one secondary methyl group. The NMR data of 8 closely showed characteristic resonances of 15-methoxy-15,16-butenolide epimers (ratio 4:1 mixture) (Table 2) (Ahmad et al., 2004; Singh et al., 1998). The major epimer showed two protons resonating at dH = 5.73 (d, J = 3.4 Hz, H15) and 6.79 (d, J = 4.4 Hz, H14), and carbon resonances at dC = 139.4, 142.1, 102.9 and 172.4 attributed to C13, C14, C15 and C16, respectively. In the ROESY spectrum cross peaks between CH319 and CH320, and CH317 and CH320 indicated that these groups were cofacial. Therefore, we could assume that the configuration of ring B was identical with that in 5–7. As to the stereochemistry of C2 and C15 four stereoisomers were possible (2R, 15R; 2R, 15S; 2S, 15R and 2S, 15S). The experimental ECD spectrum showed two negative CEs at 218 and 245 (shoulder) nm (Fig. 6). The negative CE at 245 nm resulted from the n ? p⁄ transition of the a,b-unsaturated c-lactone moiety, and the negative CE at 218 nm was likely due to the p ? p⁄ transition of an a,b-unsaturated carboxylic acid and c-lactone. We performed a conformational analysis and ECD calculation of the four possible stereoisomers. The calculated ECD spectra of the four above mentioned stereoisomers are shown in Fig. 6. The calculated spectrum of the (2S, 15S) stereoisomer showed excellent fit with the experimental data. In particular, negative CEs at 215 and 240 nm matched well with the pattern of the experimental ECD of 8. Conformational analysis using relative free energies indicated the presence of six conformers differing in orientation of the c-lactone and methoxy moieties. Slight differences between calculated and experimental spectra were due to minor differences of the theoretical and solution conformers. Taken together, the absolute configuration of 8 was 2S, 5R, 8R, 9S, 10R, 15S. Thus 8 was determined as (2S,5R,8R,9S,10R,15S)-2-b-hydroxy-15-methoxy-16-oxo-15,16dihydro-hardwickiic acid. Compound 9 (C20H32O4) showed an [MH] ion at m/z 335.2285. Comparison of NMR spectral data (Table 2) with those of floridiolic acid (Bally et al., 1976; Billet et al., 1978, 1976) showed these compounds to be identical in the side chain, but different in the decalin portion. The ROESY spectrum showed cross peaks between CH319 and CH320, and between CH317 and CH320, indicating that these groups were on the same face of the molecule. ROESY correlations H10AH6ax and H10AH8ax showed that these protons are on the same opposite side and that the H10 proton at dH = 1.34 is in the axial position. Hence, compound
Fig. 5. Minimized conformers of 6 in the gas phase using DFT at B3LYP/6-31G⁄⁄ level (left), experimental ECD spectra of 5-7, and calculated spectra of two possible stereoisomers 2R and 2S (right).
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7
Fig. 6. Minimized conformers of 8 in the gas phase using DFT at B3LYP/6-31G⁄⁄ level (left), Experimental ECD spectrum of 8, and calculated spectra of 2S,15R and 2S,15S stereoisomers.
9 had a trans decalin scaffold, in contrast to the cis decalin reported for floridiolic acid. Two stereoisomers, (5R, 8R, 9S, 10R) or (5S, 8S, 9R, 10S), were possible for compound 9, and the (5R, 8R, 9S, 10R) stereoisomer was selected for ECD calculation. Experimental and calculated ECD spectra are given in Fig. 7. The averaged ECD spectrum showed excellent fit with the experimental data, with two negative CEs at 245 and 220 nm. Thus, the absolute configuration of compound 9 established as 5R, 8R, 9S, 10R. Compound 9 was then ascertained as a neo-clerodane-15,16-diol, and particularly as (5R,8R,9S,10R)-15,16-diol-15,16-dihydro-hardwickiic acid. Only few compounds with this type of structure have been described in Salvia spp. (Merrit and Ley, 1992). Compound 10 had a molecular formula C20H28O5 (HRESI-TOFMS m/z 347.1827 for [MH], calcd. m/z 348.1937) indicating seven degrees of unsaturation. An IR absorption band at 1635 cm1 suggested the presence of an a,b-unsaturated carbonyl group. COSY and 1D TOCSY experiments showed coupling between H3 and H10, H7 and H8, and H11 and H15, establishing spin systems C3AC10, C6AC8, and C11AC15. The scaffold of 10 was assembled with the aid of HSQC and HMBC experiments (Table 2). The NMR data suggested a structure resembling that of ()-patagonic acid, but differing in ring A (Pinto et al., 2010; Rivera et al., 1988). Signals at dH = 4.40 and dC = 64.5 were consistent with the presence of an hydroxyl group at C2, and this was supported by HMBC correlations H1AC2, H3AC2, H10AC2, H2AC3, H2AC10. ROESY cross peaks between H2 and H19, CH317 and CH319, CH317 and CH320, and CH319 and CH320 indicated that these groups were cofacial. Additional ROESY cross peaks were observed for H10 and H6ax, and H10 and H8ax. Hence, they were on the opposite face of the decalin ring system, and H10 was in the axial position. Given the A/B trans fusion, two stereoisomers, (2S, 5R, 8R, 9S, 10R) and (2R, 5R, 8R, 9S, 10R), were possible. The experimental ECD spectrum of 10 showed two negative CEs at 211 and 245 nm which were due to p ? p⁄ and n ? p⁄ transitions of the a,b-unsaturated carboxylic acid and c-lactone moiety, respectively. A comparison of experimental and calculated ECD spectra for the 2S and 2R stereoisomers are shown in Fig. 8. The simulated ECD spectrum of 2S-10 showed two negative CEs at 215 and 245 nm, whereas a positive CE for the band at 215 nm was obtained for the 2R stereoisomer. Hence, compound 10 was (2S,5R,8R,9S,10R)-2-b-hydroxy-16-oxo-15,16-dihydro-hardwickiic
acid. Neo-clerodane diterpenes with a-substituted c-butenolide group like 8 and 10 have been reported for species of Salvia (Rodriguez-Hahn et al., 1994; Wu et al., 2012). Compounds 8 and 10 as well as compound 9, are described here for the first time. To the best of our knowledge this is first time that the absolute configuration of such clerodane diterpenes has been established by ECD. 2.2. Antibacterial activity of compounds Compounds 2–5 and 7–10, obtained in suitable quantities, were preliminarily tested by the qualitative Lorian method (Lorian, 2005) on a selected clinical strain belonging to the following Gram-positive species: S. aureus, S. epidermidis, Streptococcus agalactiae, E. faecalis and E. faecium, and to the following Gram-negative species: Escherichia coli and Pseudomonas aeruginosa. A Candida albicans clinical strain was also employed. The exudate mixture of S. adenophora was also assessed on the same organisms. The antimicrobial activity was evaluated by analyzing the effect on the growth of the seeded pathogens. A clear halo of inhibition was
Fig. 7. Experimental ECD spectrum of 9, and calculated spectrum of the 5R,8R,9S,10R stereoisomer.
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3. Moreover, as shown for the three oxylipins 2, 3 and 4, the antibacterial activity of compounds 9 and 10 appeared to be unrelated to the patterns of resistance of all tested pathogens to classic antibiotics. Interestingly, among those analyzed, S. epidermidis was the most susceptible species to the majority of the substances obtained from S. adenophora. 3. Conclusions
Fig. 8. Experimental ECD spectrum of 10, and calculated spectra for 2S,5R,8R,9S,10R and 2R,5R,8R,9S,10R stereoisomers.
observed particularly for compounds 2–4, 9 and 10 on different species at concentrations varying from 1000 to 125 lg/mL (data not shown). No activity was detected on the two Gram-negative organisms and on C. albicans. Interestingly, at the same concentrations, the raw extract of S. adenophora was unable to induce inhibition on the growth of any of the tested bacterial species. The MICs of the selected compounds were assessed by analyzing a total of 16 clinical strains according to standard microbiological methods (Clinical and Laboratory Standards Institute, 2008, 2010; Murray et al., 1999). Table 3 shows the results obtained. A promising antimicrobial activity was detected for compound 2 on S. aureus and S. epidermidis. Interestingly, the antibacterial potency of this substance (in terms of inhibition of growth) on both species was quite evident and very uniform particularly on S. epidermidis that displayed the lowest MIC values compared to S. aureus. Compound 3 showed a similar activity on both staphylococcal species, particularly on S. epidermidis, with MIC values even lower (16– 32 lg/mL) than those obtained with compound 2 on the same pathogen. Compound 4 was found to be active on S. agalactiae and on S. epidermidis. On S. agalactiae MIC values were lower and more uniform than on S. epidermidis, indicating a stronger antibacterial activity of this substance on the former species. Compounds 5 and 7 did not display a significant antimicrobial activity against none of the Gram-positive, Gram-negative or fungal strains analyzed here (MIC values >128 lg/mL). Hardwickic acid, whose these compounds are chemically related, was previously shown to display an antibacterial activity against Gram-positive and Gram-negative species as well as against mycobacteria (although data in the literature are sometime confusing and not univocal) (Bigham et al., 2003; Kuete et al., 2011, 2007; McChesney et al., 1991; Tene et al., 2009). Starks et al. (2010) described a moderate activity for a few similar clerodane diterpenes, obtained from Solidago virgaurea, on S. aureus, albeit on a single non-clinical strain. Compounds 8 and 10 are chemically related to patagonic acid. To our knowledge, no data about an antimicrobial activity of patagonic acid have been reported in the literature. Although compound 8 did not display a significant inhibition on the Gram-positive species tested, compound 10 displayed an interesting, although straindependent, activity specifically against S. epidermidis. A similar behavior has been displayed by compound 9 and, to our knowledge, there are no available data concerning the antibacterial activity of floridiolic acid. The antibacterial property of 9 and 10 was shown to be clearly less powerful and more strain-dependent than that described for the prostanoids, especially for compounds 2 and
Among plant metabolites, oxylipins have broad antimicrobial activities and their spectrum of action includes several microorganisms that are plant pathogens including bacteria and mycetes (Pohl et al., 2011; Prost et al., 2005; Sucharitha and Uma Maheswari Devi, 2010; Sujatha et al., 2013). Diterpenes are reported to display a wide spectrum of biological activities, including antibacterial action (Almeida et al., 2008; Kuzma et al., 2007; Porto et al., 2009a, 2009b). Diterpenoids, and particularly clerodanes, isolated from species of Salvia were also shown to possess antimicrobial activity (Fonseca et al., 2013; Kuzma et al., 2007; Radulovic et al., 2010; Stavri et al., 2009; Ulubelen, 2003; Wu et al., 2012). Our results confirm the contention that clerodanes and especially the group of oxylipins, can be considered as a source of new and interesting antimicrobial agents that deserve further study. This is particularly so in consideration of the fact that the two groups of compounds may possess different and possibly novel mechanisms of molecular action, as shown by their ability to overcome the various traits of resistance to usual antibiotics carried by the Gram-positive pathogens thus far analyzed. None of these new active molecules inhibited the two Gram-negative species tested, nor C. albicans, in agreement with previous findings showing that natural compounds obtained from several plants, while active on Gram-positive species are generally inactive on Gram-negative organisms and fungi (Bisio et al., 2008; Schito et al., 2011). This lack of activity is probably due to the peculiar structure of the external layers of both groups of microorganisms, the Gram-negatives presenting a highly selective and hydrophobic outer membrane and C. albicans, a thick cell wall where b-glucans and chitin are abundant. 4. Experimental 4.1. General experimental procedures All organic solvents used for extraction were of analytical grade and purchased from Merck (Darmstadt, Germany). Methanol, acetonitrile and formic acid used for HPLC were of HPLC-grade (Merck). Melting points are uncorrected and were measured on a Melting Point System MP50 (Mettler Toledo, Greifensee, Switzerland). Optical rotations were measured with a Perkin-Elmer 241 polarimeter (Perkin Elmer, Inc. Waltham, Massachusetts, U.S.) equipped with a sodium lamp (589 nm) and a 10 cm microcell. Silica gel 60 (Merck, 230–400 mesh) was used for column chromatography. Silica gel 60 F254 coated aluminum sheets (Merck, 20 20 cm, 0.2 mm layer thickness) were used for TLC. CHCl3ACH3OHAHCOOH (10:0.5:0.1) was used as mobile phase, and spots were detected by spraying 50% H2SO4, followed by heating. Flash chromatography was performed on a Spot Liquid Chromatography system (Armen Instrument, Saint Ave, France). Analytical and semi-preparative HPLC were carried out at room temperature using a Waters W600 pump equipped with a Rheodyne Delta 600 injector, a 2414 refractive index detector, and a 2998 photodiode array detector) (all Waters Corporation, Milford, Massachusetts, U.S.). Sample loops of 20 lL and 100 lL were used. Analytical HPLC was carried out on various C18 columns, at a flow rate of 1.0 mL/ min. Semi-preparative HPLC was carried out on various C18
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Table 3 MIC values, expressed in lg/mL and micromolarity (lM), of each isolated compound on the selected bacterial strains, compared to standard reference antibiotics. MRSA: Methicillin Resistant S. aureus, MSSA: Methicillin Susceptible S. aureus, MRSE: Methicillin Resistant S. epidermidis, MSSE: Methicillin Susceptible S. epidermidis, VRE: Vancomycin Resistant Enterococcus, Eri-R: Resistant to Erithromycin, Eri-S: Susceptible to Erithromycin, PG: Penicillin G potassium, O: Oxacillin sodium salt, A: Ampicillin sodium salt, V: Vancomycin hydrochloride, E: Erythromycin A, n.a: not active. Bacterial strains
2
3
4
9
10
5
7
8
PG
O
A
V
E
S. epidermidis 1 (MRSA) 2 (MRSA) 4 (MSSA)
32 (99) 32 (99) 32 (99)
32 (99) 16 (50) 16 (50)
32 (88) 128 (351) 64 (176)
32 (95) 128 (381) 64 (190)
32 (92) 64 (184) 125 (359)
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
1 (1) 1 (3) 2 (5)
8 (21) 8 (21) 1 (1)
– – –
– – –
– – –
S. aureus A (MRSE) MB10 (MSSE) MB24 (MRSE)
64 (199) 64 (199) 64 (199)
64 (199) 64 (199) 64 (199)
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
1 (3) 1 (1) 1 (3)
8 (21) 1 (1) 8 (21)
– – –
– – –
– – –
E. faecalis MB1 (VRE) MB3 (VRE)
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
– –
– –
32 (86) 32 (86)
16 (11) 16 (11)
– –
E. faecium 6 (VRE) MB2 (VSE)
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
n.a n.a
– –
– –
32 (86) 32 (86)
32 (22) 4 (3)
– –
S. agalactiae 149 (Eri-R) 150 (Eri-S) 155 (Eri R)
na n.a n.a
na n.a n.a
32 (88) 64 (176) 128 (351)
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
n.a n.a n.a
– – –
– – –
– – –
– – –
2 (3) 0.1 (0.2) 4 (5)
columns, at a flow rate of 2.0 mL/min. The elution mixture (helium-degassed) was composed of CH3OHAH2O at various ratios. UV spectra were recorded with an HP 8453 diode array spectrophotometer (Hewlett Packard, Palo Alto, California, U.S.). FTIR spectra were recorded as films or KBr pellets on a Perkin Elmer System 2000 instrument. A Bruker DRX-600 NMR spectrometer (Bruker, Billerica, Massachusetts, U.S.) running the XWINNMR software package was used for NMR experiments. High-resolution MS spectra were acquired in positive or in negative ion mode on a Q-TOF premier spectrometer (Waters) equipped with a electrospray ion source (ESI) and an hybrid quadrupole – W time of flight analyzer, using the following source parameters: capillary voltage 2.8 kV, sampling cone voltage 20 eV and extraction cone voltage 3 eV. A mixture of caffeine and PEG was used to perform external calibration, and quercetin was used as lock mass for accurate mass measurements. Purified compounds were dissolved in 50% acetonitrile at a final concentration of 10 ng/lL and were directly infused in the ESI at a flow rate of 5 lL/min. Acquisition was performed in continuous mode, and reported m/z values were measured by averaging at least 10 scans. 4.2. Plant material Fresh aerial parts of S. adenophora Fernald (Epling, 1940) were obtained from the Centro Regionale di Sperimentazione ed Assistenza Agricola (Albenga, Italy). The species was identified by Dr. Gemma Bramley, and a voucher specimen is deposited at the Kew Herbarium (K). 4.3. Extraction and isolation Fresh aerial parts (4.8 kg) of S. adenophora were immersed in CH2Cl2 for 20 s. After filtration, the solvent was removed under reduced pressure to afford 36.0 g of ‘‘exudate mixture’’ composed of the plant surface constituents and the content of the glandular epidermal hairs. The exudate mixture was treated with n-hexane to obtain the n-hexane-soluble and the n-hexane-insoluble fractions. The n-hexane-soluble fraction (9.8 g) was chromatographed in aliquots of 1 g on Sephadex LH-20 (53 2.5 cm; CHCl3ACH3OH 7:3 as eluent; monitoring by TLC) to afford five fractions: fraction I (3.70 g), fraction II (2.45 g), fraction III (1.98 g), fraction IV (0.19 g) and fraction V (0.06 g).
Fraction I (3.70 g) was separated by CC on silica gel (35 1.5 cm; monitoring by TLC) with mixtures of n-hexaneACHCl3 (35:65, 2.4 L; 10:90, 5.0 L; 0:100, 1.9 L) and mixtures of CHCl3ACH3OH (95:5, 1.8 L; 90:10, 1.2 L) into 22 fractions. Fraction 9 (eluted with n-hexaneACHCl3 35:65, from 2.0 to 2.2 L) was further purified by semi-preparative RP HPLC [Symmetry 300 C18, 7.8 300 mm ID, 7 lm particle size (Waters), eluents A: H2O, B: CH3OH, gradient: B 50% at time 0, B 100% at time 25 min, B 100% at time 40 min], to obtain 1 (4.6 mg). Fraction 12 (eluted with n-hexaneACHCl3 10:90, from 5.0 to 5.2 L) afforded a mixture that was further purified by semi-preparative RP HPLC [C18 column: Symmetry 300 C18, 7.8 300 mm ID, 7 lm particle size (Waters), eluents A: H2O, B: CH3OH, gradient: B 50% at time 0, B 100% at time 25 min, B 100% at time 40 min] to obtain the pure compound 2 (6.5 mg). Fraction II was chromatographed on silica gel column (35 1.5 cm; analytical TLC control) eluting with mixtures of nhexaneACHCl3 (35:65, 120 mL; 0:100, 120 mL) and mixtures of CHCl3ACH3OH (95:5, 400 mL; 90:10, 840 mL) to give in order of elution seven fractions. Elution with n-hexaneACHCl3 35:65 (from 80 to 120 mL) afforded a mixture of compounds 3 and 4, purified by semi-preparative RP HPLC [C18 column: Symmetry 300 C18, 7.8 300 mm ID, 7 lm particle size (Waters), eluents A: H2O, B: CH3OH, gradient: B 40% at time 0, B 85% at time 8 min, B 100% at time 20 min] to obtain the pure compounds 3 (6.1 mg) and 4 (9.0 mg). The n-hexane-insoluble fraction (25.93 g) was chromatographed on Sephadex LH-20 (1 g portions; 53 2.5 cm; CHCl3ACH3OH 7:3 as eluent; analytical TLC control), to give in order of elution seven fractions: fraction I (4.04 g), fraction II (0.62 g), fraction III (6.12 g), fraction IV (6.17 g), fraction V (1.24 g), fraction VI (0.44 g) and fraction VII (0.78 g). Fraction IV was chromatographed on silica gel column (55 3.5 cm; analytical TLC control) eluting with mixtures of nhexaneACHCl3 (25:75, 0.55 L; 20:80, 2.75 L; 10:90, 0.65 L; 0:100, 2.1 L) and mixtures of CHCl3ACH3OH (95:5, 3.5 L; 90:10, 0.3 L; 0:100, 0.4 L) to give in order of elution twenty-seven fractions. Elution with CHCl3 (from 1.5 to 2.1 L) afforded a mixture of ursolic and oleanolic acids, that was not further purified (120 mg), and compounds 5 and 6, purified by semi-preparative RP HPLC [C18 column: lBondapack C18, 7.8 300 mm ID, 10 lm particle size (Waters), isocratic conditions: eluent CH3OHAH2O 70:30] to obtain
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the pure compounds 5 (5.4 mg) and 6 (5.8 mg). Elution with CHCl3ACH3OH 95:5 afforded crude 7 (from 0.125 to 0.2 L), that was then purified by semi-preparative RP HPLC [C18 column: lBondapack C18, 7.8 300 mm ID, 10 lm particle size (Waters), isocratic conditions: eluent CH3OHAH2O 60:40] to obtain the pure compound 7 (170.8 mg), and a group of fractions (from 0.8 to 2.0 L) that was further chromatographed on silica gel column (35 1 cm; analytical TLC control) eluting with mixtures of n-hexaneACHCl3 (35:65, 2.0 L; 10:90, 1.0 L; 0:100, 0.3 L) and CHCl3ACH3OH (60:40, 0.4 L) to give in order of elution seventeen fractions. Fraction 11 (eluted with n-hexaneACHCl3 35:65, from 1.4 to 1.5 L) was purified by semi-preparative RP HPLC [C18 column: lBondapack C18, 7.8 300 mm ID, 10 lm particle size (Waters), isocratic conditions: eluent CH3OHAH2O 70:30] to obtain compounds 7 (6.9 mg) and 8 (4.1 mg). Fraction 14 (eluted with n-hexaneACHCl3 10:90, from 0 to 1.0 L) was purified by semi-preparative RP HPLC [C18 column: lBondapack C18, 7.8 300 mm ID, 10 lm particle size (Waters), isocratic conditions: eluent CH3OHAH2O 70:30] to obtain compound 9 (5.0 mg). Elution with CHCl3ACH3OH 90:10 (from 0 to 0.1 L) afforded crude 10, that was then purified by semi-preparative RP HPLC [C18 column: lBondapack C18, 7.8 300 mm ID, 10 lm particle size (Waters), isocratic conditions: eluent CH3OHAH2O 70:30] to obtain the pure compound 10 (24.8 mg). 4.3.1. Compound 1 Colorless gum; [a]25 D : +35.5° (c 0.3,CH3OH); ECD (MeOH, c 1.0 mM, 0.1 cm): [h]204 = 1091, [h]227 = +5082; UV/Vis kmax (CH3OH) nm (log e): 222 (3.92); IR (KBr) nmax (cm1): 3475, 3007, 2929, 2857, 1746, 1708, 1587, 1438, 1374, 1228, 1095, 1071, 1047, 888; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (positive mode) m/z: 365.2328 [M+H]+ (calcd. for C21H33O5 365.2343). ESI-MS2: m/z 323 [MCH2CO+H]+, 263 [MCH2COCH3COOH+H]+, 245 [MCH2COCH3COOHH2O+H]+, 207 [MCH2COCH3COOHCH2CHCHO+H]+. 4.3.2. Compound 2 Colorless gum; [a]25 D : +29.9° (c 0.14,CH3OH); ECD (MeOH, c 1.4 mM, 0.1 cm): [h]222 = +7167; UV/Vis kmax (CH3OH) nm (log e): 222 (4.14); IR (KBr) nmax (cm1): 3474, 2928, 2856, 1738, 1706, 1587, 1456, 1352, 1211, 1108; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (positive mode) m/z: 323.2229 [M+H]+ (calcd. for C19H30O4Na 323.2222); ESI-MS2: m/z 305 [MH2O+H]+, 263 [MCH3OCOH+H]+, 245 [MCH3OCOHH2O+H]+, 207 [MCH3COOHCH2CHCHO+H]+. 4.3.3. Compound 3 Colorless gum; [a]25 D : +60.0° (c 0.19,CH3OH); ECD (MeOH, c 1.5 mM, 0.1 cm): [h]218 = +6415; UV/Vis kmax (CH3OH) nm (log e): 221 (3.58); IR (KBr) nmax (cm1): 3463, 2928, 2856, 1738, 1706, 1588, 1456, 1352, 1211, 1109; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (positive mode) m/z: 323.2224 [M+H]+ (calcd. for C19H31O4 323.2222); ESI-MS2: m/z 305 [MH2O+H]+, 263 [MCH3OCOH+H]+, 245 [MCH3OCOHH2O+H]+, 207 [MCH3COOHCH2CHCHO+H]+. 4.3.4. Compound 4 Colorless gum; [a]25 D : +107.3° (c 0.18,CH3OH); ECD (MeOH, c 1.7 mM, 0.1 cm): [h]222 = +8083; UV/Vis kmax (CH3OH) nm (log e): 220 (3.24); IR (KBr) nmax (cm1): 3489, 2930, 2857, 1747, 1709, 1587, 1438, 1374, 1230, 1096, 1072, 1048; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (positive mode) m/z: 365.2396 [M+H]+ (calcd. for C21H33O5 365.2343). ESI-MS2: m/z 323 [MCH2CO+H]+, 263 [MCH2COCH3COOH+H]+, 207 [MCH2COCH3COOHCH2CHCHO+H]+.
4.3.5. Compound 5 Powder; MP: 79–80 °C; [a]25 D : 52.0° (c 1.00, CHCl3); ECD (MeOH, c 0.4 mM, 0.1 cm): [h]211 = 93,235, [h]248 = 16,103 (shoulder); UV/Vis kmax (CH3OH) nm (log e): 208 (3.77); IR (KBr) mmax (cm1): 3425, 2959, 2929, 2648, 1740, 1694, 1452, 1372, 1237, 1024, 873, 775, 600; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (negative mode): m/z 373.4598 [MH], (calcd. for C22H29O5:373.4626); ESI-MS2: m/z 329 [MCO2H], 313 [MCH3COOHH], 269 [MCO2CH3COOHH]. 4.3.6. Compound 6 Powder; MP: 112–114 °C; [a]25 D : 84.4° (c 0.15,CH3OH); ECD (MeOH, c 1.4 mM, 0.1 cm): [h]211 = 19,816, [h]248 = 6435 (shoulder); UV/Vis kmax (CH3OH) nm (log e): 206 (3.42); IR (KBr) mmax (cm1): 3434, 2957, 2917, 2871, 1736, 1638, 1504, 1454, 1382, 1249, 1022, 870, 767, 631; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (negative mode): m/z 359.2217 [MH], (calcd for C22H31O4: 359.2222); ESI-MS2: m/z 315 [MCO2H], 313 [MC2H5OHH], 253 [MCO2C2H5OHH]. 4.3.7. Compound 7 Powder; MP: 114–116 °C; [a]25 D : 220.5° (c 0.08,CH3OH); ECD (MeOH, c 0.9 mM, 0.1 cm): [h]211 = 20,748, [h]248 = 5889 (shoulder); HRESI-TOFMS (negative mode): m/z 331.1920 [MH], (calcd. for C20H27O4: 331.1909). 4.3.8. Compound 8 Powder; MP: 113–114 °C; [a]25 D : 51.1° (c 0.24,CH3OH); ECD (MeOH, c 0.8 mM, 0.1 cm): [h]211 = 18,058, [h]248 = 5141 (shoulder); UV/Vis kmax (CH3OH) nm (log e): 207 (3.97); IR (KBr) mmax (cm1): 3423, 2958, 2923, 2632, 1770, 1690, 1452, 1385, 1254, 1211, 1083, 1023, 935, 846, 769, 636; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (negative mode): m/z 377.2003 [MH], (calcd. for C21H29O6: 377.1964); ESI-MS2: m/z 345 [MCH3OHH], 333 [MCO2H], 315 [MCO2H2OH], 301 [MCH3OHCO2H]. 4.3.9. Compound 9 Powder; MP: 112–114 °C; [a]25 D : 34.7° (c 0.23,CH3OH); ECD (MeOH, c 0.9 mM, 0.1 cm): [h]242 = 8352; UV/Vis kmax (CH3OH) nm (log e): 225 (3.65); IR (KBr) mmax (cm1): 3406, 2957, 2922, 2630, 1709, 1463, 1384, 1225, 976, 804, 687; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (negative mode): m/z 335.2225 [M H], (calcd. for C20H31O4: 335.2222); ESI-MS2: m/z 317 [MH2OH], 291 [MCO2H], 273 [MH2OCO2H], 255 [M2(H2O)CO2H], 243 [MH2OCO2CH2OH]. 4.3.10. Compound 10 Powder; MP: 94–96 °C; [a]23 D : 254.64° (c 0.19,CHCl3); ECD (MeOH, c 0.4 mM, 0.1 cm): [h]211 = 50,686, [h]248 = 14,830 (shoulder); UV/Vis kmax (CH3OH) nm (log e): 209 (4.23); IR (KBr) mmax (cm1): 3433, 2947, 2924, 2640, 1746, 1678, 1636, 1384, 1259, 1214, 1084, 1022, 930, 842, 769, 703; 1H NMR and 13C NMR: Tables 1 and 2; HRESI-TOFMS (negative mode): m/z 347.1827 [MH], (calcd. for C20H27O5: 347.1858); ESI-MS2: m/z 329 [MH2OH], 303 [MCO2H], 285 [MCO2H2OH], 259 [MCO2CO2H], 243 [MCO2CH3OCHOH]. 4.4. Computational methods Conformational analysis of 1–10 was performed with Schrödinger MacroModel 9.1 (Schrödinger, LLC, Portland, OR) employing the OPLS2005 (optimized potential for liquid simulations) force field in H2O. Conformers within a 2 kcal/mol energy window from the global minimum were selected for geometrical optimization and energy calculation applying DFT with the Becke’s nonlocal
Please cite this article in press as: Bisio, A., et al. Antibacterial compounds from Salvia adenophora Fernald (Lamiaceae). Phytochemistry (2014), http:// dx.doi.org/10.1016/j.phytochem.2014.10.033
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three parameter exchange and correlation functional, and the Lee– Yang–Parr correlation functional level (B3LYP) using the 6-31G⁄⁄ basis set in the gas phase with the Gaussian 09 program package (Frisch et al., 2009). Vibrational evaluation was done at the same level to confirm minima. Excitation energy (denoted by wavelength in nm), rotatory strength dipole velocity (Rvel), and dipole length (Rlen) were calculated in CH3OH by TD-DFT/ B3LYP/631G⁄⁄, using the SCRF method, with the CPCM model. ECD curves were obtained on the basis of rotator strengths with a half-band of 0.25 eV using SpecDis v1.61. (Bruhn et al., 2013). The ECD spectra were calculated from the spectra of individual conformers according to their contribution to Boltzmann-weighting. 4.5. Antimicrobial experiments 4.5.1. Microorganisms Thirteen strains of five Gram-positive species (S. aureus, S. epidermidis, S. agalactiae, E. faecium and E. faecalis), two clinical strains of Gram-negative species (E. coli and P. aeruginosa), and a C. albicans strain, previously isolated from different clinical specimens and identified according to standard procedures (Murray et al., 1999) were used. A total of 16 strains were employed in this study. Of these, 1 was a methicillin-susceptible S. aureus (MSSA), 2 methicillin-resistant and multidrug-resistant S. aureus (MRSA), 1 methicillin-susceptible S. epidermidis (MSSE) and 2 methicillin-resistant S. epidermidis (MRSE), 1 vancomycin-susceptible (VAN-S) E. faecalis, 1 vancomycin-resistant (VAN-R) E. faecalis, 1 VAN-S E. faecium and 1 VAN-R E. faecium, 1 erithromycin-susceptible S. agalactiae group B (SAGB) and 2 erithromycin-resistant S. agalactiae group B, one strain of E. coli and P. aeruginosa, and 1 C. albicans clinical strain. Our definition of multidrug-resistant organisms, in keeping with the literature (Clinical and Laboratory Standards Institute, 2008, 2010) is ‘‘a strain that displays at least 3 resistance traits to chemically unrelated antibiotics’’. 4.5.2. Active compounds and control antibiotics Sterile stock solutions in dimethyl sulfoxide (DMSO) of S. adenophora pure compounds and exudate mixture were prepared. More diluted solutions were obtained using the appropriate media required for the experiments. Control antibiotics (Ampicillin sodium salt, Erythromycin A, Oxacillin sodium salt, Penicillin G potassium, Vancomycin hydrochloride) (Sigma–Aldrich, Saint Louis, MO, U.S.) were diluted in the appropriate media as specified by Clinical and Laboratory Standards Institute, 2010. 4.5.3. Preliminary evaluation of antimicrobial activity To evaluate the antimicrobial activities of compounds and exudate mixture we followed a procedure detailed by Lorian (2005), which entails the use of a replicator in order to spot the liquid onto the surface of an appropriately seeded agar plate. Selected bacterial and fungal strains were grown overnight at 37 °C on Mueller Hinton plates (MH) (Biolife, Milan, Italy) or on Columbia agar, supplemented with 5% sheep blood (Becton, Dickinson and Company, Maryland, USA). After incubation, 5–7 colonies of each strain were suspended in sterile 0.1 M phosphate buffer (PB). The density of the suspensions was determined in a McFarland nephelometer (Dalynn Biological Inc.) and adjusted in order to obtain a final concentration of about 1.5 108 CFU/mL. Aliquots of 0.01 mL of the bacterial or fungal suspensions were spread on MH agar plates, Columbia agar plates, for S. agalactiae, or on Sabouraud Agar (Oxoid) for C. albicans, and 10.0 lL of the exudate mixture at several 2-fold dilutions in DMSO (ranging from 1 to 64 mg/L) were spotted on the surface of the plate. The plates were then incubated overnight at 37 °C for bacteria and at 22 °C for 48 h for the yeast, and the diameter of the resulting zones of inhibition were analyzed. For control purposes pure DMSO, as well as several twofold
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dilutions, were also spotted on the same plate. No inhibition of growth was observed at any concentrations tested. 4.5.4. Susceptibility testing activity Minimum inhibitory concentrations (MICs) were determined following the microdilution procedure (Clinical and Laboratory Standards Institute, 2008, 2010) using as a test medium for bacteria MH or, when needed, cation-supplemented (Ca2+ and Mg2+) MH broth, or cation-adjusted MH broth with 2% lysed horse blood. For antifungal tests, RPMI-1640 medium with L-glutamine, and buffered with MOPS buffer was used. Briefly, overnight cultures of bacteria were diluted to yield a final concentration of 5 105 cells/mL for bacteria, and 0.5–2.5 103 CFU/mL for fungi. Two fold serial dilutions of drugs, ranging from 0.015 to 128 lg/mL, were prepared in the required broth in 96-well plates. An equal volume of bacterial or fungal inoculum was added to each well on the micro titer plate containing 100 lL of the appropriately diluted compounds. After 24 h of incubation at 37 °C for bacteria, and at 22 °C for 48 h for the yeast, the lowest concentration of each compound that prevented a visible growth was recorded as the MIC. All MICs were obtained in triplicate. Variations in the values never exceeded a single twofold dilution. MICs reported in Table 3 represent two identical results obtained in the 3 testings. The MICs of the reference antibiotics against each bacterial species were calculated by following the guidelines proposed by EUCAST, 2014 (The European Committee on Antimicrobial Susceptibility Testing, 2014). Each test was performed in triplicates. Acknowledgement Università degli Studi di Genova – Italy – PRA project 2013 is kindly acknowledged. 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. 2014.10.033. References Ahmad, V.U., Farooq, U., Abbaskhan, A., Hussain, J., Abbasi, M.A., Nawaz, S.A., Choudhary, M.I., 2004. Four new diterpenoids from Ballota limbata. Helv. Chim. Acta 87, 682–689. Ainai, T., Matsuumi, M., Kobayashi, Y., 2003. Efficient total synthesis of 12-oxo-PDA and OPC-8:0. J. Org. Chem. 68, 7825–7832. Almeida, L.S.B., Murata, R.M., Yatsuda, R., dos Santos, M.H., Nagem, T.J., Alencar, S.M., Koo, H., Rosalen, P.L., 2008. Antimicrobial activity of Rheedia brasiliensis and 7-epiclusianone against Streptococcus mutans. Phytomedicine 15, 886–891. Andersson, M.X., Hamberg, M., Kourtchenko, O., Brunnström, Å., McPhail, K.L., Gerwick, W.H., Göbel, C., Feussner, I., Ellerström, M., 2006. Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana: formation of a novel oxophytodienoic acid-containing galactolipid, Arabidopside E. J. Biol. Chem. 281, 31528–31537. Baertschi, S.W., Ingram, C.D., Harris, T.M., Brash, A.R., 1988. Absolute configuration of cis-12-oxophytodienoic acid of flaxseed: implications for the mechanism of biosynthesis from the 13(S)-hydroperoxide of linolenic acid. Biochemistry-US 27, 18–24. Bally, R., Billet, D., Durgeat, M., Heitz, S., 1976. Constituants d’Evodia floribunda Baker – II – 2éme Partie. Confirmation par rayons X de la structure de l’ester methylique de l’acide floridiolique. Tetrahedron Lett. 32, 2777–2778. Bigham, A.K., Munro, T.A., Rizzacasa, M.A., Robins-Browne, R.M., 2003. Divinatorins A–C, new neoclerodane diterpenoids from the controlled sage Salvia divinorum. J. Nat. Prod. 66, 1242–1244. Billet, D., Durgeat, M., Heitz, S., Brouard, J.P., 1978. Diterpenes d’Evodia floribunda Baker III. J. Chem. Res. (S), 110–111. Billet, D., Durgeat, M., Heitz, S., Brouard, J.P., Ahond, A., 1976. Constituants d’Evodia floribunda Baker – II – 1ère Partie. L’acide floridiolique, nouveau diterpene de type clerodane. Tetrahedron Lett. 32, 2773–2776. Bisio, A., Romussi, G., Russo, E., Cafaggi, S., Schito, A.M., Repetto, B., De Tommasi, N., 2008. Antimicrobial activity of the ornamental species Salvia corrugata, a potential new crop for extractive purposes. J. Agric. Food Chem. 56, 10468– 10472.
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