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Abietanes from Plectranthus grandidentatus and P. hereroensis against methicillin- and vancomycin-resistant bacteria.

Abstract

The antimicrobial activity of 10 natural abietanes isolated from Plectranthus grandidentatus and P. hereroensis acetonic extract was evaluated against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). The results revealed that the most active diterpenes were coleon U (1), 7[alpha]-acetoxy-6[beta]-hydroxyroyleanone (2) and horminone (3). Minimum inhibitory concentration (MIC) values ranging 0.98-15.63 [micro]g/ml were obtained for MRSA clinical strains, and MIC values of 15.63 and 31.25 [micro]g/ml were obtained for VRE clinical strains. Some structure-activity relationships are emphasized.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Plectranthus sp.; Abietanes; Antimicrobial; Anti-MRSA activity; Coleon U; Royleanones

Introduction

Lamiaceae is an Angiospermae family rich in diterpenes (with quite a skeleton diversity) (Alvarenga et al., 2001), as well as in phenolic compounds (Grayer et al., 2003). Plectranthus is a well-represented genus of the Nepetoideae subfamily (Lamiaceae), in eastern and southern tropical Africa flora. It is of interest to explore the potential medicinal and economic uses of Plectranthus, for the reasons that some are African medicinal plants (Ferreira et al., 1997; Matu and van Staden, 2003; Willis et al., 2003) with potential use in primary healthcare, and that this genus is related to Salvia, Coleus and Rabdosia genera (Alvarenga et al., 2001) used in ethnomedicine all over the world. In addition, the bioactive secondary metabolites found in these plants are structurally related, as is the case with oxidized abietane diterpenes (Alvarenga et al., 2001; Abdel-Mogib et al., 2002; Ulubelen, 2003). Due to the multiple pharmacological activities described for these oxidized abietane metabolites, it is thinkable to use them as models for the search of further bioactive drugs.

The widely spread infections caused by bacteria resistant to multiple antibiotics have become an important health problem. Both methicillin-resistant Staphylococcus aureus (MRSA), one of the causes of nosocomial infections (Sousa and Lencastre, 2003), and vancomycin-resistant Enterococcus faecalis (VRE) (Nelson et al., 2000) are increasingly found in hospitals worldwide. Therefore, the search for new antimicrobial compounds active against multidrug-resistant bacteria has great relevance. An antibacterial study carried out on oxidized abietanes (Yang et al., 2001) showed that some were active against MRSA and VRE strains. In our continuing search for bioactive compounds from Plectranthus, several royleanone and coleon abietanes with antimicrobial (Batista et al., 1995; Teixeira et al., 1997), antitumoral (Marques et al., 2002) and T- and B-lymphocytes were found suppressing proliferation activities (Cerqueira et al., 2004).

Now we present an antimicrobial study of 10 abietanes isolated from Plectranthus hereroensis and P. grandidentatus against six MRSA and two VRE clinical strains. The relationship between the structure of these diterpenes and its antimicrobial activity is analysed (Fig. 1 and Table 1).

Materials and methods

Plant material, extraction and isolation

Aerial parts and roots of cultivated (from original seeds) P. grandidentatus Gurke (voucher specimen, C. Marques S/No, deposited in LISC, Centro de Botanica, Instituto de Investigacao Cientifica e Tropical-MCT) and P. hereroensis Engl. (voucher specimen, no. 160382, deposited in the Herbarium of Instituto Botanico, Universidade de Lisboa) were extracted with acetone, followed mainly by chromatographic isolation of pure abietane diterpenes as previously described (Marques et al., 2002; Teixeira et al., 1997; Batista et al., 1994, 1995, 1996; Marques et al., 2002).

[FIGURE 1 OMITTED]

Tested compounds

The selected abietanes from P. grandidentatus (whole plant) were coleon U (1, 20 mg, 7.8 x [10.sup.-4]% w/w on dry plant) (Marques et al., 2002), 7[alpha]-acetoxy-6[beta]-hydroxyr-oyleanone (2, 410mg, 1.6 x [10.sup.-2]%) (Teixeira et al., 1997; Marques et al., 2002), horminone (3, 18mg, 7.0 x [10.sup.-4]%) (Teixeira et al., 1997), 6[beta]-hydroxyroyleanone (4, 6mg, 2.3 x [10.sup.-4]%) (Teixeira et al., 1997), 6[beta], 7[alpha]-dihydroxyroyleanone (5, 10.6mg, 4.1 x [10.sup.-4]%) (Marques et al., 2002), royleanone (6, 10mg, 3.9 x [10.sup.-4]%) (Teixeira et al., 1997) and 6,7-dehydroroyleanone (7, 11 mg, 4.3 x [10.sup.-4]%) (Teixeira et al., 1997). The abietanes selected from P. hereroensis roots were horminone (3, 32mg, 1.5 x [10.sup.-2]%) (Batista et al., 1994), 7[alpha], 12-dihydroxy-17(15[right arrow]16)abeo-abieta-8,12,16-triene-11,14-dione (8, 11 mg, 5.0 x [10.sup.-3]%) (Batista et al., 1994), 16-acetoxy-7[alpha], 12-dihydroxy-8, 12-abietadiene-11, 14-dione (9, 30mg, 1.4 x [10.sup.-2]%) (Batista et al., 1995) and that from the aerial parts of P. hereroensis was 3[beta]-acetoxy-6[beta], 7[alpha], 12-trihydroxy-17(15[right arrow]16), 18(4[right arrow]3)-bisabeo-abieta-4(19),8,12,16-tetraene-11,14-dione (10, 13 mg, 9.5 x [10.sup.-4]%) (Batista et al., 1996). The structures of these known compounds were established by comparing their spectral data with those in the literature and/or with authentic samples.

Antimicrobial assay

Six acquired MRSA and two VRE isolates were selected for the present study from strain collections of the Microbiology Laboratory, Faculty of Pharmacy, Lisbon.

The quantitative antimicrobial evaluation was performed by the determination of minimum inhibitory concentration (MIC) values on pure compounds against the clinical strains. MIC values were determined using the twofold serial broth microdilution assay (NCCLS, 2000). The compounds were dissolved in DMSO with a graded concentration ranging 250-0.49 [micro]g/ml with a Muller-Hinton broth medium for bacteria. The antibacterial activity of the solvent was evaluated and control antibiotics were included. The MIC value was taken as the lowest concentration of compound that inhibited the growth of the test organisms after 24 h of incubation at 37[degrees] (Table 1).

Results

A previous evaluation of antimicrobial activities against standard Gram-negative and positive bacteria and yeast strains (Salmonella typhimurium ATCC 43971, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, S. aureus ATCC 25923 and Candida albicans CIP 3153A) has been done for some abietanes (Marques et al., 2002; Teixeira et al., 1997; Batista et al., 1995, 1994). Thus, we also tested coleon U (1) and 6[beta],7[alpha]-dihydroxyroyleanone (5) for those standard microrganisms. Diterpene 1 revealed activity against P. aeruginosa, E. coli and C. albicans (MIC value 31.25 [micro]g/ml) and potent activity against S. aureus (MIC value 7.81 [micro]g/ml), while compound 5 showed only activity for S. aureus (MIC value 31.25 [micro]g/ml). The majority of the assayed diterpenes showed activity against S. aureus (Gram-positive bacteria). These former data prompted us to test the activity of 10 natural abietanes (1-10) against six MRSA and two VRE clinical strains.

The MIC values presented in Table 1 show that coleon U (1), 7[alpha]-acetoxy-6[beta]-hydroxyroyleanone (2) and horminone (3) were the most active compounds against MRSA (MIC values 0.98-15.63 [micro]g/ml), with better MIC values than positive control oxacillin. They also show moderate activity against VRE strains (MIC values of 15.63 and 31.25 [micro]g/ml). Coleon U (1) showed potent activity against five of the MRSA strains (MIC value of 0.98 [micro]g/ml). Diterpene 2 showed good activity (MIC values ranging from 3.91 to 15.63 [micro]g/ml) against the six MRSA strains, and so did horminone (3) (MIC values ranging from 7.81 to 15.63 [micro]g/ml). Amongst the remaining compounds, abietanes 8 and 9 showed higher activity (MICs of 15.63-62.50 [micro]g/ml) for MRSA strains than the compounds 4-7 and 10 (Table 1). Abietanes 5, 8 and 9 were more active against VRE strains (MIC values of 31.25-62.50[micro]g/1) (Table 1).

Discussion

Comparing MIC values of abietanes 1-10, some structure-activity relationships may be suggested. It must be emphasized that the potent antibacterial activity of coleon U (1), the only abietane with the more oxygenated and dehydrogenated chromophoric system through rings B and C (a diosphenolic oxohydroquinone, characteristic of a coleon-type abietane).

The results of the other abietanes suggest that antibacterial activity against MRSA and VRE depends on the presence of a 12-hydroxy-p-benzoquinone moiety in ring C (characteristic of a royleanone-type abietane) simultaneously with an oxidized B ring at the C-6/C-7 positions (royleanones 2-5, 8, 9). Moreover, if the oxidation is at C-7 (3), as well as if there are simultaneously a 6[beta]-hydroxyl and a 7[alpha]-acetoxyl substitution pattern (2), the activity is higher. An exclusive oxidation at C-6 (abietane 4) or a simultaneous hydroxylation at C-6 and C-7 (compound 5) were translated into poor antibacterial activity. The presence of the acetoxy group at C-7 (2) is translated into better antimicrobial activity (see MICs for 3-6), perhaps because this group confers partial lipophilic character allied to the concomitant presence of the hydrophilic moiety (-COO-). Royleanones 6 and 7, without oxygen functions in the B ring, had no activity against all MRSA and VRE strains.

The similar lipophilic character and the electronic structure on the quininoid C ring, together with the oxygenated groups of ring B at C-7, probably point to the molecular and cellular mechanism of the antibacterial activity of these abietanes being close to that imputed to horminone (3, mechanism consequent to a protonophoric activity and to a quelation ability with [Mg.sup.2+]ion) (Nicolas et al., 2003; Spiridonov et al., 2003).

If MIC values for royleanones 8 (with a C-13 allylic side chain) and 9 (with a C-13 acetoxyisopropyl side chain) are compared with the MIC values of the analogous horminone (3), it is clear that the C-13 isopropyl side chain of 3 confers higher activity against the clinical strains assayed, as already referred for Gram-positive antibacterial activity (Yang et al., 2001; Batista et al., 1995; Moujir et al., 1993; San Feliciano et al., 1993). Rearranged royleanone 10 showed poor activity due, at least, to the simultaneous presence of two hydroxyl groups on C-6/C-7 and a C-13 allylic side chain.

It is noteworthy that coleon U (1) and 7[alpha]-acetoxy-6[beta]-hydroxyroyleanone (2) present not only the highest antibacterial activity against MRSA clinical strains, but also have interesting antitumoral activities (Marques et al., 2002) and strongly suppress the proliferation of T and B-lymphocytes (Cerqueira et al., 2004).

Acknowledgements

The authors thank FCT (I & D No 8/94), POCTI (QCA III) and FEDER for financial support and for a Ph.D. Grant to one of us (Praxis XXI/BD/18046/98). We thank Prof J. Nascimento for his helpful collaboration. We are grateful to Dr. Eurico S. Martins and Dr. Belo Correia for plant identification and to the National Botanical Garden, Kirstenbosh, Claremont, South Africa for supplying seed material.

References

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C. Gaspar-Marques (a), P. Rijo (a), M.F. Simoes (a,*), M.A. Duarte (b), B. Rodriguez (c)

(a) Faculdade de Farmacia, Universidade de Lisboa, CECF, Lisboa, Portugal

(b) Lab. Microbiologia, Faculdade de Farmacia, Universidade de Lisboa, Lisboa, Portugal

(c) Instituto de Quimica Organica, CSIC, Madrid, Spain

Received 3 May 2004; accepted 14 June 2005

*Corresponding author. Tel.: +352 217946200; fax: 352 217946400.

E-mail address: fasimoes@ff.ul.pt (M.F. Simoes).
Table 1. MIC values ([micro]g/ml) of abietanes against methicillin-
resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus (VRE)

 MRSA
Compounds (a) 1 2

Coleon U (1) 0.98 0.98
7[alpha]-Acetoxy-6[beta]-hydroxyroyleanone (2) 7.81 7.81
Horminone (3) 7.81 15.63
6[beta]-Hydroxyroyleanone (4) >125 (b) >125 (b)
6[beta],7[alpha]-Dihydroxyroyleanone (5) >125 (b) >125 (b)
Royleanone (6) >125 (b) >125 (b)
6,7-Dehydroroyleanone (7) >125 (b) >125 (b)
7[alpha],12-Dihydroxy-17(15[right arrow]16)- 15.63 62.50
abeoabieta-8,12,16-triene-11,14-dione (8)
16-Acetoxy-7[alpha],12-dihydroxy-8,12-abietadiene- 62.50 62.50
11,14-dione (9)
3[alpha]-Acetoxy-3[alpha],7[beta],12-trihydroxy- >125 (b) 62.50
17(15[right arrow]16),18(4[right arrow]3)-bisabeo-
abieta-4(19),8,12,16-tetraene-11,14-dione (10)
Oxacillin 15.63 15.63
Vancomycin nt (c) nt (c)

 MRSA
Compounds (a) 3 4

Coleon U (1) 0.98 0.98
7[alpha]-Acetoxy-6[beta]-hydroxyroyleanone (2) 3.91 3.91
Horminone (3) 7.81 15.63
6[beta]-Hydroxyroyleanone (4) 62.50 62.50
6[beta],7[alpha]-Dihydroxyroyleanone (5) 62.50 62.50
Royleanone (6) >125 (b) >125 (b)
6,7-Dehydroroyleanone (7) >125 (b) >125 (b)
7[alpha],12-Dihydroxy-17(15[right arrow]16)- 31.25 31.25
abeoabieta-8,12,16-triene-11,14-dione (8)
16-Acetoxy-7[alpha],12-dihydroxy-8,12-abietadiene- 31.25 31.25
11,14-dione (9)
3[alpha]-Acetoxy-3[alpha],7[beta],12-trihydroxy- 62.50 >125 (b)
17(15[right arrow]16),18(4[right arrow]3)-bisabeo-
abieta-4(19),8,12,16-tetraene-11,14-dione (10)
Oxacillin 15.63 15.63
Vancomycin nt (c) nt (c)

 MRSA
Compounds (a) 5 6

Coleon U (1) 3.91 0.98
7[alpha]-Acetoxy-6[beta]-hydroxyroyleanone (2) 15.63 7.81
Horminone (3) 15.63 15.63
6[beta]-Hydroxyroyleanone (4) >125 (b) >125 (b)
6[beta],7[alpha]-Dihydroxyroyleanone (5) >125 (b) >125 (b)
Royleanone (6) >125 (b) >125 (b)
6,7-Dehydroroyleanone (7) >125 (b) >125 (b)
7[alpha],12-Dihydroxy-17(15[right arrow]16)- 62.50 62.50
abeoabieta-8,12,16-triene-11,14-dione (8)
16-Acetoxy-7[alpha],12-dihydroxy-8,12-abietadiene- 62.50 62.50
11,14-dione (9)
3[alpha]-Acetoxy-3[alpha],7[beta],12-trihydroxy- >125 (b) >125 (b)
17(15[right arrow]16),18(4[right arrow]3)-bisabeo-
abieta-4(19),8,12,16-tetraene-11,14-dione (10)
Oxacillin 15.63 15.63
Vancomycin nt (c) nt (c)

 VRE
Compounds (a) le 2e

Coleon U (1) 31.25 31.25
7[alpha]-Acetoxy-6[beta]-hydroxyroyleanone (2) 15.63 31.25
Horminone (3) 15.63 31.25
6[beta]-Hydroxyroyleanone (4) >125 (b) >125 (b)
6[beta],7[alpha]-Dihydroxyroyleanone (5) 62.50 31.25
Royleanone (6) >125 (b) >125 (b)
6,7-Dehydroroyleanone (7) >125 (b) >125 (b)
7[alpha],12-Dihydroxy-17(15[right arrow]16)- 62.50 31.25
abeoabieta-8,12,16-triene-11,14-dione (8)
16-Acetoxy-7[alpha],12-dihydroxy-8,12-abietadiene- 31.25 31.25
11,14-dione (9)
3[alpha]-Acetoxy-3[alpha],7[beta],12-trihydroxy- 62.50 62.50
17(15[right arrow]16),18(4[right arrow]3)-bisabeo-
abieta-4(19),8,12,16-tetraene-11,14-dione (10)
Oxacillin nt (c) nt (c)
Vancomycin 250 125

(a) The compounds were dissolved in a mixture of DMSO: broth medium
(1:1).
(b) In this concentration, the observed inhibition is equal to a control
with the same solvent mixture but without a tested compound.
(c) nt--not tested.
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Author:Gaspar-Marques, C.; Rijo, P.; Simoes, M.F.; Duarte, M.A.; Rodriguez, B.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Mar 1, 2006
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