Two new diterpenoids from Plectranthus species

Phytochemistry Letters 3 (2010) 221–225

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Two new diterpenoids from Plectranthus species Maria Fa´tima Simo˜es a,*, Patrı´cia Rijo a, Aida Duarte a, Diana Barbosa a, Diogo Matias a, Joana Delgado a, Na´dia Cirilo a, Benjamı´n Rodrı´guez b a b

Faculdade de Farma´cia da Universidade de Lisboa, iMed.UL, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal Instituto de Quı´mica Orga´nica, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 June 2010 Received in revised form 25 July 2010 Accepted 9 August 2010 Available online 21 August 2010

Two new diterpenoids, ent-7a-acetoxy-15-beyeren-18-oic acid and (13S,15S)-6b,7a,12a,19-tetrahydroxy-13b,16-cyclo-8-abietene-11,14-dione, have been isolated from Plectranthus saccatus and Plectranthus porcatus, respectively, and their structures were established by 1D and 2D NMR spectroscopic studies. The new diterpenes showed no activity against Gram-negative bacteria and Candida albicans (yeast strain). Among Gram-positive bacteria, the lower MIC value was 62.50 mg/ml for the abietane derivative against Staphylococcus aureus ATCC 6538. ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Plectranthus saccatus Plectranthus porcatus Lamiaceae Diterpenes Beyerane 13,16-Cycloabietane Biological activity

1. Introduction The genus Plectranthus (family Lamiaceae) comprises ca. 350 species distributed over Tropical Africa, Asia and Australia. Many Plectranthus species are plants of economical, ornamental and medicinal interest, and with a vast array of ethnobotanical uses (Lukhoba et al., 2006). Several species are used in African traditional medicine for the treatment, among others, of wounds (Githinji and Kokwaro, 1993) and gastro-intestinal disorders (Kokwaro, 1993), for the alleviation of respiratory conditions (Boily and Van Puyvelde, 1986), as antimicrobial agents (Matu and Van Staden, 2003) and for malaria (Githinji and Kokwaro, 1993; Schlage et al., 2000). Moreover, many Plectranthus plants have been tested worldwide for a variety of biological actions and, among others, they have an antiproliferation effect on human lymphocytes (Cerqueira et al., 2004), are good insect repellents (Omolo et al., 2004) and can control insect feeding habits (Wellsow et al., 2006). Several Plectranthus plants have been studied chemically and a large number of abietane, phyllocladane, kaurane, clerodane and labdane diterpenoids have been isolated from them, together with oleanane, ursane and lupane triterpenoids, aristolane and aromadendrane sesquiterpenes, flavonoids and long-chain alkylphenols

* Corresponding author. Tel.: +351 217946400; fax: +351 217946470. E-mail address: [email protected] (M.F. Simo˜es).

(Abdel-Mogib et al., 2002; Gaspar-Marques et al., 2004; Rijo et al., 2002). In continuation of our studies on biologically active diterpenoids from Plectranthus species (Cerqueira et al., 2004; GasparMarques et al., 2004, 2006, 2008; Rijo et al., 2002, 2010), we have now investigated Plectranthus porcatus Van Jaarsv. & P. Winter (Winter and Van Jaarsveld, 2005), a species that has not hitherto been studied either chemically or pharmacologically, and Plectranthus saccatus Benth., a plant from which two beyerane diterpenoids (3 and 4, Fig. 1) possessing insect antifeedant activity against Spodoptera littoralis have recently been isolated (Wellsow et al., 2006). This paper describes the structure elucidation of the new diterpenes 1 and 2 (Fig. 1) found in P. saccatus and P. porcatus, respectively, as well as the results of their antimicrobial activity against reference bacteria and yeast. 2. Results and discussion The new diterpenoid (1) isolated from an acetone extract of the aerial parts of P. saccatus (see Section 3.3) was a colourless crystalline solid, for which a molecular formula of C22H32O4 was established by HRESIMS. The 1H and 13C NMR spectra of 1 (Table 1) were very similar to those of 3, a 15-beyerene diterpenoid previously isolated from the same plant (Wellsow et al., 2006). The 1 H and 13C NMR spectra of 1 showed signals, among others, for a carboxylic acid function (dC 184.1, q), a (Z)-1,2-disubstituted olefin (dH 5.53 d and 5.55 dd, 1H each, Jvic = 5.8 Hz) linked directly to

1874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2010.08.002

[(Fig._1)TD$IG]

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M.F. Simo˜es et al. / Phytochemistry Letters 3 (2010) 221–225 Table 1 1 H and 13C NMR spectroscopic data for compound 1.a Position

Fig. 1. Structures of the diterpenoids.

Configurationb

dC

1

38.0, CH2

2

17.6, CH2

3

36.8, CH2

4 5 6

46.7, qC 42.4, CH 27.4, CH2

7 8 9 10 11

76.0, 52.9, 48.1, 36.6, 19.6,

12

32.7, CH2

13 14

43.9, qC 56.6, CH2

15 16 17 18 19 20 OAc

132.1, CH 138.1, CH 24.7, CH3 184.1, qC 16.4, CH3 14.9, CH3 170.8, qC 21.1, CH3

CH qC CH qC CH2

a b a b a b –

b a b a –

b –

a b a b –

a b b b a a –

dH 1.64 1.02 1.53 1.53 1.64 1.72 – 2.24 1.72 1.49 4.76 – 1.45 – 1.28 1.53 1.26 1.26 – 1.44 1.26 5.53 5.55 1.01 – 1.15 0.79 – 2.04

c

m mc mc mc mc td dd mc mc t mc mc mc mc mc dd d d ddd s s s

J (H,H)

Hz

1a,1b 1a,2a 1a,2b 1b,2a 1b,2b 2a,2b 2a,3a 2a,3b 2b,3a 2b,3b 3a,3b 5b,6a 5b,6b 6a,6b 6a,7a 6b,7a 9b,11a 9b,11b 11a,11b 11a,12a 11a,12b 11b,12a 11b,12b 12a,12b 12a,14ae 14a,14b 15,16

c c c c c c c 14.7 c 2.5 14.7 12.8 1.5 c 2.7 2.7 c c c c c c c c 2.1 9.1 5.8

s

a

quaternary carbon atoms (see footnote d in Table 1) and an acetoxyl group (dH 2.04, 3H, s; dC 170.8, q, and 21.1, CH3). This acetoxyl substituent must be axially attached to a secondary carbon atom (dC 76.0, CH; dH 4.76, 1H, t, J e;a0 ¼ J e;e0 ¼ 2:7 Hz) which was placed between a fully substituted carbon and a methylene group. The observed HMBC connectivities between the carbonyl carbon of the acetate (d 170.8) and the proton at d 4.76, and between this proton and C-5, C-6, C-8, C-9, C-14 and C-15 (d 42.4, 27.4, 52.9, 48.1, 56.6 and 132.1, respectively), established that the acetoxyl substituent of 1 was at the 7b-position (ent-7a). The HMBC spectrum of 1 showed long-range connectivities between the carboxylic carbon (dC 184.1) and the protons at d 2.24 (H-5), 1.72 and 1.64 (H2-3), and 1.15 (quaternary methyl group), whereas the methyl protons at d 1.15 were HMBC correlated with C-3, C-4, C-5 and with the carboxylic carbon (d 36.8, 46.7, 42.4 and 184.1, respectively). These results established that both a carboxylic function and a quaternary methyl group at dH 1.15 (dC 16.4) were attached to the C-4 position (dC 46.7, q) of the beyerane skeleton. From the 13C NMR chemical shifts of the carboxylic carbon (d 184.1) and its geminal methyl group (d 16.4) it was evident (Bruno et al., 1986; Hussein et al., 1999; Hussein and Rodrı´guez, 2000; Wellsow et al., 2006) that in 1 the carboxyl group was an equatorial substituent (18-position) and, consequently, the methyl group was axially oriented (19-position), with opposite stereochemistry to that in 3 (Wellsow et al., 2006). 1D NOESY experiments confirmed the relative stereochemistry depicted in 1 for this new diterpenoid and allowed the complete assignment of the overlapped proton signals shown in Table 1. Irradiation at d 5.54 (both H-15 and H-16 protons) caused NOEs in H-6a, H-7a, H-11a, H-14a, Me-17 and Me-20, whereas the signal of the H-15 proton, among others, was strongly enhanced when the H-7a proton signal (d 4.76) was irradiated. Moreover, on irradiation at d 0.79 (Me-20) NOEs were observed in H-1a, H-2a, H-6a, H-11a, H-15 and, more important, in Me-19. The absolute configuration of 1 was not ascertained, although on biogenetic grounds (Wellsow et al., 2006) we assume that this compound belongs to the enantio series, like the majority of the beyerane-type diterpenes isolated from natural sources and whose

In CDCl3 solution, at 500 MHz (1H) and 125 MHz (13C). Chemical shifts are referenced to the solvent signals (residual CHCl3 at d 7.26 for protons and d 77.00 for carbons). All these assignments were in agreement with COSY, HSQC and HMBC spectra. b For clarity, the a- or b-configuration for a substituent indicates that it is placed, respectively, below or above the plane of the formula depicted for 1. However, since we assume that this compound belong to the enantio series, those configurations should be described more rigorously as ent-b or ent-a, indicating that the substituent is placed, respectively, below or above the plane of the depicted structure. c Overlapped or partially overlapped signals; d values were deduced from the HSQC spectrum. d This proton is long-range coupled (J = 1.0 Hz) with one of the 12-methylene protons, as was revealed by the 1H–1H COSY spectrum. e This is a W-type coupling.

absolute stereochemistry has been rigorously determined (Connolly and Hill, 1991; Jefferies et al., 1963; McMillan and Beale, 1999). From all the above data 1 was characterized as ent-7aacetoxy-15-beyeren-18-oic acid. It is of interest to comment that the co-occurrence in the same plant of diterpenes oxidized at the C-18 (1) or C-19 (3 and 4) (Wellsow et al., 2006) positions is quite rare (Connolly and Hill, 1991), and only a few cases have been reported hitherto (Connolly and Hill, 1991; Matloubi-Moghadam et al., 1984). Repeated chromatographic processes on the acetone extract of the aerial parts of P. porcatus (see Section 3.3) allowed the isolation of 2, which possessed a molecular formula of C20H28O6 (HRESIMS). The 1H NMR spectrum of 2 (Table 2) was almost identical to that of spirocoleon 13 (5), a compound previously isolated (Matloubi-Moghadam et al., 1984) from Coleus somaliensis S. Moore. In fact, the only observed differences were consistent with the absence in the former of the 7-O-acetyl group of the latter [2: dH-7b 4.63 dd, J7b,6a = 1.9 Hz, J7b,7a-OH = 4.9 Hz; a doublet (J = 1.9 Hz) after addition of D2O. 5: dH-7b = 5.75 d, J7b,6a = 2 Hz; d 1.97, 3H, s, 7a-OAc (Matloubi-Moghadam et al., 1984)]. Moreover, the 13C NMR spectrum of 2 (Table 2) was in complete agreement with those reported (Ru¨edi et al., 1983) for other spirocoleons possessing the same oxidation pattern and an identical stereochemistry.

M.F. Simo˜es et al. / Phytochemistry Letters 3 (2010) 221–225 Table 2 1 H and 13C NMR spectroscopic data for compound 2.a

dC

Configuration

dH

1

38.8, CH2

2

20.1, CH2

3

40.3, CH2

a b a b a b

4 5 6

39.8, qC 50.3, CH 70.3, CH

7

67.2, CH

1.29 2.05 1.50 1.79 1.26 1.55 – 1.72 4.23 4.99 4.63 4.26 – – – – 3.82 5.12 – – 2.02 1.08 0.92 1.26 1.09 4.24 3.33 4.98 1.74

Position

–

a a b-OH b a-OH

8 9 10 11 12

142.5, qC 156.2, qC 40.1, qC 200.3, qC 78.6, CH

– – – –

13 14 15 16

37.5, qC 198.0, qC 21.6, CH 27.3, CH2

– –

17 18 19

14.2, CH3 29.4, CH3 68.7, CH2

20

22.7, CH3

b a-OH a b-A (pro-S) b-B (pro-R) b a b-A b-B b-OH b

td mb ddddd ddddd ddd br d d mc dd dd dd

d dd

mb dd dd d s dd dd br dd s

J (H,H)

Hz

1a,1b 1a,2a 1a,2b 1b,2a 1b,2b 2a,2b 2a,3a 2a,3b 2b,3a 2b,3b 3a,3b 5a,6a 6a,6b-OHd 6a,7b 7b,7a-OHd 12b,12a-OHd 15,16A (pro-S) 15,16B (pro-R) 15,17 16A,16B 19A,19B 19A,19-OHd 19B,19-OHd

13.5 3.6 13.5 3.1 3.4 14.1 3.6 3.1 13.5 3.4 14.0 1.6 1.8 1.9 4.9 5.5 8.5 6.7 6.1 3.7 11.1 4.4 5.5

In acetone-d6 solution, at 500 MHz (1H) and 125 MHz (13C). Chemical shifts are referenced to the solvent signals (d 2.05 for protons and d 30.5 for carbons). All the assignments were in agreement with COSY, HSQC and HMBC spectra. b These signals appeared overlapped with the solvent signals and their d values were deduced from the HSQC spectrum. In CD3OD solution (see Section 3.4), these protons appeared at d 2.07 (1H, ddd, J1b,1a = 13.0 Hz, J1b,2a = 3.0 Hz, J1b,2b = 3.7 Hz, H1b) and d 2.11 (1H, ddq, J15,16A = 8.6 Hz, J15,16B = 6.8 Hz, J15,17 = 6.1 Hz, H-15a). c Partially overlapped signal. d These signals and their respective couplings (3J) disappeared after addition of D2O.

223

6538 with MIC value of 62.50 mg/ml. This strain is the reference bacterium for assays of disinfectant efficacy, most of these products interfere with cytoplasmic membrane of the prokaryote cell, so the preferential activity of 2 in S. aureus ATCC 6538 could indicate the affinity of this compound for the cellular membrane. 3. Experimental 3.1. General experimental procedures Melting-points were uncorrected and measured on a Kofler block. Optical rotations were determined in CHCl3 or MeOH solutions (Perkin-Elmer 241 MC polarimeter). IR was taken in KBr (Perkin-Elmer Spectrum One spectrophotometer). 1H and 13C NMR spectra (at 500 and 125 MHz, respectively) were acquired in CDCl3 (1), acetone-d6 (2) or CD3OD (2) solution at room temperature (Varian SYSTEM 500 MHz spectrometer). NMR chemical shifts are reported with respect to the residual CHCl3 (d 7.26), acetone-d6 (d 2.05) or CD3OD (d 3.30) signals for the 1H NMR spectra, and to the solvent signals (CDCl3: d 77.0, acetone-d6: d 30.5) for the 13C NMR spectra. EIMS was obtained in the positive mode (Hewlett-Packard Model 5973 instrument, 70 eV). HRESIMS was obtained in an Agilent 6520 Accurate-Mass QTOF LC/MS apparatus. Merck 5554 Kieselgel 60 F254 sheets were used for thin-layer chromatographic (TLC) analysis. Light petroleum (b.p. 50–70 8C) was used for column chromatography.

a

The stereochemistry at the C-12, C-13 and C-15 chiral centers of 2 was established by comparing the 1H and 13C NMR chemical shifts and JH,H values corresponding to the C-12–C-17 structural fragment (Table 2) with those reported for 5 (Matloubi-Moghadam et al., 1984) and other related spirocoleons (Ku¨nzle et al., 1987; Ru¨edi et al., 1983; Schmid et al., 1982). These values for 2 were identical with those of compounds with a (12R,13S,15S)-configuration and very different from those of the other diastereoisomers (Ku¨nzle et al., 1987; Matloubi-Moghadam et al., 1984; Ru¨edi et al., 1983; Schmid et al., 1982). Compound 2 showed a positive sign for its specific rotation (½a20 D þ 218:8) like 5 and other spirocoleons previously found in plants belonging to the Plectranthus and Coleus genera (Ku¨nzle et al., 1987; Matloubi-Moghadam et al., 1984; Ru¨edi et al., 1983; Schmid et al., 1982) and whose absolute stereochemistry has been rigorously established. For this reason, and also on biogenetic grounds, we assume that 2 possesses a normal-abietane absolute configuration. Thus, this new diterpenoid is (13S,15S)6b,7a,12a,19-tetrahydroxy-13b,16-cyclo-8-abietene-11,14dione (2). Both diterpenoids (1 and 2) showed no activity against Gramnegative bacteria (Escherichia coli and Pseudomonas aeruginosa) and Candida albicans. Compound 1 exhibited MIC value of 125 mg/ ml against Mycobacterium smegmatis and Staphylococcus aureus strains. A literature survey did not account for antimicrobial beyeranes. Though many royleanone-type abietanes are described as antibacterial and antimycobacterial agents (Gaspar-Marques et al., 2006; Rijo et al., 2010; Yang et al., 2001), the cycloabietane 2 showed the same results as 1 with the exception for S. aureus ATCC

3.2. Plant materials P. saccatus Benth. and P. porcatus Van Jaarsv. & P. Winter, grown in ‘‘Parque Botaˆnico da Tapada da Ajuda’’ (AJUDA) from cuttings provided by the Kirstenbosch National Botanical Gardens, South Africa, were collected in July–October 2007, and in June and September 2008, respectively. Voucher specimens (P. saccatus, LISI 835/2007; P. porcatus, LISI 109/2008) were deposited in the Herbarium ‘‘Joa˜o de Carvalho e Vasconcellos’’ of the ‘‘Instituto Superior de Agronomia’’, Lisbon (LISI). 3.3. Extraction and isolation The air-dried and powdered aerial parts of P. saccatus (100 g) were extracted with Me2CO (8 0.6 l) at room temperature for 10 days. Filtration and evaporation of the solvent (under vacuum, 40 8C) yielded a residue (6.89 g, 6.89% of dry plant material), which was subjected to column chromatography over silica gel (Merck 9385, 75 g), using mixtures of light petroleum–EtOAc 1:0 to 0:1 and MeOH as eluents. The fraction eluted with light petroleum– EtOAc 1:1 (2.419 g) was filtered through a pad of charcoal and celite (1:1) to eliminate chlorophylls. The resulting residue was fractionated by column chromatography (silica gel, Merck 7734, 8 g) eluting with light petroleum–EtOAc 7:3, giving 47 mg of impure 1 that, after recrystallization from light petroleum, yielded 24 mg (0.024% of the plant material) of pure beyerane 1. The air-dried and powdered aerial parts of P. porcatus (99 g) were extracted with Me2CO (5 0.2 l) at room temperature for 5 days. Filtration and evaporation of the solvent (under vacuum, 40 8C) yielded 8.46 g of crude extract (8.54%). This extract was subjected to column chromatography over silica gel (Merck 9385, 180 g), using mixtures of increasing polarity of light petroleum– EtOAc 1:0 to 0:1 as eluents. The fraction eluted with light petroleum–EtOAc 3:7 (1.403 g) was further fractionated by column chromatography (Merck 7734, 20 g; light petroleum– EtOAc 3:7 as eluent) giving 0.286 g of impure 2 that, after crystallization from MeOH–pentane, yielded 9.37 mg (0.00946%, on dry plant material) of pure abietane 2.

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3.4. Compound characterization 3.4.1. ent-7a-Acetoxy-15-beyeren-18-oic acid (1) Colourless rectangular prisms (light petroleum), m.p. 240– 242 8C; ½a20 D þ 43:5 (CHCl3, c 0.299); IR (KBr), nmax 3413, 2949, 2866, 1728, 1695, 1452, 1387, 1267, 1246, 1184, 1022, 977, 858, 756, 745 cm1; EIMS: m/z (relative intensity, %) 360 [M]+ (1), 318 (2), 300 (42), 285 (17), 272 (100), 255 (12), 239 (13), 185 (14), 157 (19), 146 (65), 131 (29), 118 (48), 105 (34), 91 (30), 79 (22), 67 (12), 55 (17); HRESIMS: m/z 360.2261 [MH] (calcd. 360.2262 for C22H31O4). For 1H and 13C NMR spectra, see Table 1. 3.4.2. (13S,15S)-6b,7a,12a,19-tetrahydroxy-13b,16-cyclo-8abietene-11,14-dione (2) Yellowish fine needles (MeOH–pentane), m.p. 200–203 8C, decomposition; ½a20 D þ 218:8 (MeOH, c 0.637); IR (KBr), nmax 3547, 3407, 3346, 3030, 2956, 2927, 2868, 1700, 1670, 1458, 1372, 1317, 1282, 1215, 1091, 1020, 909, 737 cm1; EIMS: m/z (relative intensity, %) 364 [M]+ (12), 346 (36), 328 (100), 313 (31), 300 (73), 285 (72), 271 (72), 229 (55), 217 (72), 201 (50), 189 (42), 177 (40), 123 (36), 109 (39), 95 (50), 91 (58), 77 (41), 67 (33), 55 (66); HRESIMS: m/z 365.1941 [M+H]+ (calcd. 365.1957 for C20H29O6). For 1H and 13C NMR spectra in acetone-d6, see Table 2. 1H NMR (500 MHz, CD3OD): d 4.63 (1H, d, J7b,6a = 2.5 Hz, H-7b), 4.21 (1H, br s, W1/2 = 6 Hz, H-6a), 4.19 (1H, d, J19A,19B = 11.1 Hz, HA-19), 3.75 (1H, s, H-12b), 3.29 (1H, d, J19B,19A = 11.1 Hz, HB-19), 2.11 (1H, ddq, J15,16A = 8.6 Hz, J15,16B = 6.8 Hz, J15,17 = 6.1 Hz, H-15a), 2.07 (1H, ddd, J1b,1a = 13.0 Hz, J1b,2a = 3.0 Hz, J1b,2b = 3.7 Hz, H-1b), 1.81 (1H, qt, J2b,2a = J2b,1a = J2b,3a = 14.2 Hz, J2b,1b = J2b,3b = 3.7 Hz, H2b), 1.70 (3H, s, Me-20), 1.68 (1H, br s, W1/2 = 2.2 Hz, H-5a), 1.56 (1H, ddd, J3b,3a = 14.2 Hz, J3b,2a = 3.0 Hz, J3b,2b = 3.7 Hz, H-3b), 1.53 (1H, dtt, J2a,2b = 14.2 Hz, J2a,1a = J2a,3a = 3.7 Hz, J2a,1b = J2a,3b = 3.0 Hz, H-2a), 1.29 (1H, ddd, J1a,1b = 13.0 Hz, J1a,2a = 3.7 Hz, J1a,2b = 14.2 Hz, H-1a), 1.26 (1H, m, overlapped signal, H-3a), 1.26 (3H, d, J17,15 = 6.1 Hz, Me-17), 1.15 (1H, dd, J16A,16B = 3.7 Hz, J16A,15 = 8.6 Hz, HA-16), 1.09 (3H, s, Me-18), 0.93 (1H, dd, J16B,16A = 3.7 Hz, J16B,15 = 6.8 Hz, HB-16). 3.5. Biological evaluation 3.5.1. Microdilution method The minimum inhibitory concentrations (MICs) values of compounds, against the test strains, were performed by means of the twofold serial broth microdilution assay (CLSI, 2006). The compounds, dissolved in DMSO, were diluted at concentrations ranging from 500 to 0.49 mg/ml, with a Mu¨eller-Hinton broth medium for bacteria, and a Sabouraud broth medium for the yeast strain. The antimicrobial activity of the solvent was evaluated. Ampicillin (AMP), ketoconazole (KTC), methicillin (MET), norfloxacin (NOR), rifampicin (RIF) and vancomycin (VAN) were used as control antibiotics. The MIC values were taken as the lowest concentration of the compound that inhibited the growth of the microorganisms, after 24 h of incubation at 37 8C, and are presented in mg/ml unities. The bacterial growth was measured with an Absorvance Microplate Reader set to 630 nm (ELX808TM – BioteK). Assays were carried out in triplicate for each tested microorganism. 3.5.2. Microbial strains C. albicans ATCC 10231 (MICKTC = 1.95), E. coli ATCC 25922 (MICNOR < 0.49), P. aeruginosa ATCC 27853 (MICNOR < 0.49), M. smegmatis ATCC 607 (MICRIF < 0.49), S. aureus ATCC 25923 (MICMET = 0.98, MICVAN = 1.95), S. aureus ATCC 6538 (MICRIF < 0.49), S. aureus ATCC 43866 (MICMET = 1.95, MICVAN = 3.91), S. aureus ATCC 700699 (MICMET = 15.63, MICVAN = 7.81), S. aureus CIP 106760 (MRSA, MICMET > 250, MICVAN = 3.91), S. aureus FFHB 29593 (MRSA,

MICMET > 250, MICVAN = 1.95), S. epidermis ATCC 12228 (MICRIF < 0.49), Enterococcus faecalis ATCC 51299 (low VRE, MICAMP < 0.49, MICVAN = 62.50), E. faecalis FFHB 427483 (MICAMP < 0.49, MICVAN = 1.95), E. hirae ATCC 10541 (MICAMP < 0.49). MRSA means methicillin resistant S. aureus strains and low VRE is a low level vancomycin resistant Enterococcus strain. FFHB species were identified and deposited on the Microbiology Laboratory of the Faculty of Pharmacy, Lisbon University, from clinical isolates of Hospital do Barreiro. Acknowledgements We are grateful to Ernst van Jaarsveld, horticulturist, Kirstenbosch National Botanic Gardens (South Africa), for providing cuttings of Plectranthus plants, and to Teresa Vasconcelos, researcher LISI (Lisbon), for the growing of the plants. This work was supported by funds from the Portuguese ‘‘Fundac¸a˜o para a Cieˆncia e a Tecnologia’’ (FCT-MCES to iMed.UL and PhD Grant No. SFRH/BD/19250/2004) and from the Spanish ‘‘Ministerio de Ciencia e Innovacio´n’’, Grant No. CTQ2009-10343, and ‘‘Consejerı´a de Educacio´n de la Comunidad de Madrid’’ (Project CAPOTE-S2009/ PPQ-1752). References Abdel-Mogib, M., Albar, H.A., Batterjee, S.M., 2002. Chemistry of the genus Plectranthus. Molecules 7, 271–301. Boily, Y., Van Puyvelde, L.J., 1986. Screening of medicinal plants of Rwanda (Central Africa) for anti-microbial activity. J. Ethnopharmacol. 16, 1–13. Bruno, M., Savona, G., Ferna´ndez-Gadea, F., Rodrı´guez, B., 1986. Diterpenoids from Salvia greggii. Phytochemistry 25, 475–477. Cerqueira, F., Cordeiro-Da-Silva, A., Gaspar-Marques, C., Simo˜es, F., Pinto, M.M., Nascimento, M.S.J., 2004. Effect of abietane diterpenes from Plectranthus grandidentatus on T- and B-lymphocyte proliferation. Bioorg. Med. Chem. 12, 217– 223. Clinical and Laboratory Standards Institute, 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved standard. CLSI publication no. M100-S16, 7th ed. CLSI, Wayne, PA. Connolly, J.D., Hill, R.A., 1991. Dictionary of Terpenoids, vol. 2. Chapman and Hall, London, pp. 956–962. Gaspar-Marques, C., Simo˜es, M.F., Rodrı´guez, B., 2004. Further labdane and kaurane diterpenoids and other constituents from Plectranthus fruticosus. J. Nat. Prod. 67, 614–621. Gaspar-Marques, C., Rijo, P., Simo˜es, M.F., Duarte, M.A., Rodrı´guez, B., 2006. Abietanes from Plectranthus grandidentatus and P. hereroensis against methicillinand vancomycin-resistant bacteria. Phytomedicine 13, 267–271. Gaspar-Marques, C., Simo˜es, M.F., Valdeira, M.L., Rodrı´guez, B., 2008. Terpenoids and phenolics from Plectranthus strigosus, bioactivity screening. Nat. Prod. Res. 22, 167–177. Githinji, C.W., Kokwaro, J.O., 1993. Ethnomedicinal study of major species in the family Labiatae from Kenya. J. Ethnopharmacol. 39, 197–203. Hussein, A.A., Rodrı´guez, B., Martı´nez-Alca´zar, M.P., Cano, F.H., 1999. Diterpenoids from Lycopus europaeus and Nepeta septemcrenata: revised structures and new isopimarane derivatives. Tetrahedron 55, 7375–7388. Hussein, A.A., Rodrı´guez, B., 2000. Isopimarane diterpenoids from Lycopus europaeus. J. Nat. Prod. 63, 419–421. Jefferies, P.R., Rosich, R.S., White, D.E., 1963. Absolute configuration of beyerol. Tetrahedron Lett. 4, 1793–1799. Kokwaro, J.O., 1993. Medicinal Plants of East Africa. Kenya Literature Bureau, Nairobi. Ku¨nzle, J.M., Ru¨edi, P., Eugster, C.H., 1987. Isolation and structure elucidation of 36 diterpenoids from leaf-glands of Plectranthus edulis (Vatke) T. T. Aye. Helv. Chim. Acta 70, 1911–1929. Lukhoba, C.W., Simmonds, M.S.J., Paton, A.J., 2006. Plectranthus: a review of ethnobotanical uses. J. Ethnopharmacol. 103, 1–24. Matloubi-Moghadam, F., Ru¨edi, P., Eugster, C.H., 1984. Novel coleons and royleanones from Coleus somaliensis S. Moore. Helv. Chim. Acta 67, 201–208. Matu, E.M., Van Staden, J.H., 2003. Anti-bacterial and anti-inflammatory activities of some plants used for medicinal purposes in Kenya. J. Ethnopharmacol. 87, 35–41. McMillan, J., Beale, M.H., 1999. Diterpene biosynthesis. In: Barton, D., Nakanishi, K., Meth-Cohn, O. (Eds.), Comprehensive Natural Products Chemistry, vol. 2. Elsevier, Oxford, pp. 217–243. Omolo, M.O., Okinyo, D., Ndiege, I.O., Lwande, W., Hassanali, A., 2004. Repellency of essential oils of some Kenyan plants against Anopheles gambiae. Phytochemistry 65, 2797–2802. Rijo, P., Gaspar-Marques, C., Simo˜es, M.F., Duarte, A., Apreda-Rojas, M.C., Cano, F.H., Rodrı´guez, B., 2002. Neoclerodane and labdane diterpenoids from Plectranthus ornatus. J. Nat. Prod. 65, 1387–1390.

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