Fitoterapia 80 (2009) 432–436
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Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Antimicrobial ent-pimarane diterpenes from Viguiera arenaria against Gram-positive bacteria Thiago Souza Porto a, Niege A.J.C. Furtado b, Vladimir C.G. Heleno a, Carlos H.G. Martins a, Fernando B. Da Costa b, Marcela E. Severiano a, Aline N. Silva a, Rodrigo C.S. Veneziani a,⁎, Sérgio R. Ambrósio a,⁎ a b
Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, Franca, SP, Brazil Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil
a r t i c l e
i n f o
Article history: Received 25 March 2009 Accepted in revised form 27 May 2009 Available online 12 June 2009 Keywords: Pimarane diterpenes Antimicrobial activity Viguiera arenaria Gram-positive bacteria
a b s t r a c t The dichloromethane crude extract from the roots of Viguiera arenaria (VaDRE) has been employed in an antimicrobial screening against several bacteria responsible for human pathologies. The main diterpenes isolated from this extract, as well as two semi-synthetic pimarane derivatives, were also investigated for the pathogens that were significantly inhibited by the extract (MIC values lower than 100 μg mL− 1). The VaDRE extract was significantly active only against Gram-positive microorganisms. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); PA sodium salt; ent-8(14),15-pimaradien-3β-ol; ent-15-pimarene-8β,19-diol; and ent-8(14),15-pimaradien-3β-acetoxy displayed the highest antibacterial activities (MIC values lower than 10 μg mL− 1 for most pathogens). In conclusion, our results suggest that pimaranes are an important class of natural products for further investigations in the search of new antibacterial agents. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Bacterial infectious diseases are a serious public health problem worldwide [1–3]. The increase in bacterial resistance and the rapid emergence of new infections have drastically decreased the efficacy of the drugs employed in the treatment of pathologies caused by certain microorganisms [1]. The rise in community-acquired methicillin-resistant Staphylococcus aureus (MRSA); Streptococcus pneumoniae resistant to penicillins, macrolides, and fluoroquinolones; vancomycin-resistant Enterococci; and the multidrug-resistant strain of Mycobacterium tuberculosis illustrate the urgent need for new antibiotics and/or novel approaches concerning clinical treatment options [2,4,5]. The great reservoir of chemical structures obtained from higher plants continues to provide new and important leads
⁎ Corresponding authors. Tel./fax: +55 16 37118878. E-mail addresses:
[email protected] (R.C.S. Veneziani),
[email protected] (S.R. Ambrósio). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.06.003
against several pharmacological targets [6–8]. Although secondary plant metabolites are considered a rich source of new potential therapeutic agents, just a small number of pharmacological evaluations have been performed [9–11]. Studies aiming at the investigation of the biological activity of natural compounds provide an important step toward the discovery and development of novel drugs [12,13]. Diterpenoids are a large class of plant-derived natural products exhibiting a wide spectrum of important biological activities [9,12]. Many reports have shown that this class of secondary metabolites displays antiparasitic effects [14–16], inhibits vascular smooth muscle contraction [9,17,18], has analgesic and anti-inflammatory activities [19–21], among other functions. Moreover, this class of compounds has been reported to display significant antimicrobial activity against Gram-positive and Gram-negative bacteria and yeasts [4,22–24]. Our research group has concentrated efforts on the discovery of new natural compounds extracted from Brazilian plants that display antibacterial activity against oral pathogens. We have demonstrated that some pimarane-type diterpenes isolated from Viguiera arenaria are able to inhibit
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the growth of Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis, and Lactobacillus casei with very promising minimal inhibitory concentration (MIC) values (ranging from 2 to 10 μg mL− 1). We have also pointed out that these metabolites may be potentially employed in the further development of natural anti-caries agents [25,26]. In view of these results, we have decided to perform an antimicrobial screening against a panel of bacteria responsible for several pathologies using the dichloromethane crude extract from the roots of V. arenaria (VaDRE), a well known source of pimarane diterpenes [27]. We have also evaluated the antibacterial activity of the main diterpenes isolated from this extract and two semi-synthetic pimarane derivatives for the pathogens which were significantly inhibited by the initial crude extract [28]. 2. Experimental 2.1. General NMR spectra were performed on a Bruker DPX 400 spectrometer (400 MHz for 1H and 100 MHz for 13C). Samples were dissolved in CDCl3 or CD3OD, with TMS as internal reference; the chemical shifts are given in ppm. 2.2. Plant material Viguiera arenaria Baker (Asteraceae) was collected by F.B. Da Costa from the vicinity of the Washington Luís highway (km 223, 22°10 S, 47°59 W, SP, Brazil, in March 1999). The plant material was identified by J. N. Nakajima (Universidade Federal de Uberlândia, MG, Brazil) and E. E. Schilling (University of Tennessee, TN, USA). A voucher specimen (FBC 60) was deposited under the code SPFR 4006 in the herbarium of the Departamento de Biologia; Faculdade de
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Filosofia, Ciências e Letras de Ribeirão Preto;Universidade de São Paulo; SP; Brazil. 2.3. Extraction and isolation An aliquot (8.0 g) of the dichloromethane crude extract from the roots of V. arenaria (VaDRE), which was prepared in our laboratory [27], was suspended in a CH3OH/H2O solution (9:1 v/v) and partitioned with n-hexane and dichloromethane (DCM). The n-hexane (2.0 g) and DCM fractions (1.0 g) were re-fractioned using several chromatographic techniques, such as vacuum liquid chromatography, flash chromatography, and preparative thin-layer chromatography, as well as recrystallization with methanol, as previously described [14,27]. These procedures furnished 100 mg ent-8 (14),15-pimaradiene (1); 200 mg ent-pimara-8(14),15-dien19-oic acid (PA; 2); 7 mg 7-keto-ent-pimara-8(14),15-dien19-oic acid (3); 100 mg ent-8(14),15-pimaradien-3β-ol (4); 20 mg ent-8(14),15-pimaradiene-3β,19-diol (5); 5 mg ent15-pimarene-8β,19-diol (6); and 20 mg 7β-hydroxy-entpimara-8(14),15-dien-19-oic acid (7). 2.4. Semi-synthetic derivatives Compound 8 (37 mg) was obtained from 4 (50 mg) as described by Da Costa et al. [16]. Compound 9 (40 mg) was prepared from 2 (50 mg) according to Daló et al. [29]. The chemical structures of these compounds were established by 1 H- and 13C-NMR spectral data analysis and comparison with literature data [26]. 2.5. Purity of the evaluated diterpenes The purity of each diterpene (1–9; Fig. 1) was estimated by thin-layer chromatography using different solvent systems.
Fig. 1. Chemical structures of the pimarane diterpenes from Viguiera arenaria.
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They were also submitted to 1H and 13C NMR spectral data analysis, which indicated that the purity grade fell between 95 and 98% for each compound. 2.6. Antimicrobial assays The MIC values (the lowest concentration of the compound capable of inhibiting microorganism growth) of the extract and the pure diterpenes were determined in triplicate by using the microdilution broth method in 96-well microplates [25,26]. The strains of the following microorganisms were used in the present work: Kocuria rhizophila (ATCC 9341); Streptococcus pyogenes (ATCC 19615); S. pneumoniae (ATCC 6305); Enterococcus hirae (ATCC 10541); S. aureus (ATCC 9144); S. aureus (ATCC 6538); S. aureus (ATCC 29213); Bacillus subtilis (ATCC 6051); Bacillus cereus (ATCC 14579); Streptococcus dysgalactiae (ATCC 9926); Streptococcus agalactiae (ATCC 27591); Staphylococcus epidermidis (ATCC 12228); Enterobacter faecalis (ATCC 19433); Enterococcus aerogenes (ATCC 13048); Pseudomonas aeruginosa (ATCC 27853); Escherichia coli (ATCC 14948); Proteus mirabilis (ATCC 29906); Morganella morganii (ATCC 25829); Citrobacter freundii (ATCC 8090); and Shigella flexneri (ATCC 12022). The samples were dissolved in DMSO (dimethyl sulfoxide) at 1 mg mL− 1, followed by dilution in tryptic soy broth; concentrations ranging from 100 to 1 μg∙mL− 1 were achieved. The final DMSO content was 4% (v/v), and this solution was used as negative control. The inoculum was adjusted for each organism, to yield a cell concentration of 5 × 105 colony forming units (CFU).mL− 1, according to previously standardization by the National Committee for Clinical Laboratory Standards. One inoculated well was included, to allow control of the adequacy of the broth for organism growth. One noninoculated well, free of antimicrobial agent, was also included, to ensure medium sterility. Vancomycin hydrochloride and streptomycin sulfate were used as positive control for Grampositive and Gram-negative bacteria, respectively. The microplates (96-wells) were sealed with plastic film and incubated at 37 °C for 24 h. After that, resazurin (30 μL) in aqueous solution (0.02%) was added to the microplates, to indicate microorganism viability [25,26,30,31]. 3. Results and discussion The chemical structures of the diterpenoids evaluated in this work are presented in Fig. 1. The spectral data of all compounds are in agreement with those previously reported in the literature: 1, 2, 3, 6, and 7 [32,33]; 4 [34]; 5 [35]; 8 and 9 [26]. Our work was initially concerned with the antimicrobial screening of the dichloromethane extract from the roots of V. arenaria, a Brazilian plant rich in pimarane-type diterpenes [27], against several Gram-positive and Gram-negative bacteria. Table 1 shows that this extract has very promising MIC values (lower than 100 μg mL− 1) [28] for most Grampositive pathogens. These results have led us to evaluate the antibacterial activity of nine pimarane diterpenes: the seven main constituents of the extract (1–7), and two other obtained by semi-synthetic means (8 and 9). Among all the evaluated diterpenes, compounds 2, 4, 6, 8, and 9 displayed the
highest antibacterial activity (Table 1), with MIC values lower than 10 μg mL− 1 for several pathogens. Compounds 6 and 8 were less active against S. aureus strains, with MIC values higher than 50 μg mL− 1. The compounds were tested only against the microorganisms that were significantly responsive during the antimicrobial screening performed with the plant extract. The plant kingdom is an important source of new and effective antimicrobial agents, because plants have the ability to produce natural products for chemical defense against microorganisms present in their own environment [1,4,36]. As part of this scenario, the present paper points out the significant antimicrobial activity displayed by ent-pimarane diterpenes isolated from V. arenaria against several Grampositive bacteria. Some previous reports have described the effective antimicrobial activity displayed by several pimarane diterpenes. For instance, Dettrakul et al. [37] demonstrated that diaporthein B strongly inhibits M. tuberculosis growth with a MIC value of 3.1 μg mL− 1; Thongnest et al. [38] showed that two pimarane diterpenes from Kaempferia marginata, namely sandaracopimaradien-1α-ol and 2α-acetoxysandaracopimaradien-1α-ol, display significant antituberculous activity against M. tuberculosis H37Ra, with MIC values of 25 and 50 μg/mL, respectively; the compound ent-isopimara-9 (11),15-diene-19-ol was found to be very active against MRSA, with an MIC value of 2 μg mL− 1 [39]. Our research group has recently demonstrated that the compounds entpimara-8(14),15-dien-19-oic acid (PA); ent-8(14),15-pimaradien-3β-ol; ent-15-pimarene-8β,19-diol; ent-8(14),15pimaradien-3β-acetoxy; and the PA sodium salt derivative are very effective anticariogenic agents. Therefore, research involving the ent-pimarane diterpenes could pave the way for the discovery of new natural compounds that could be employed in the development of oral care products. According to Gibbons [36] and Ríos and Recio [28], some considerations must be borne in mind in the study of antimicrobial assays of plant extracts, essential oils, and compounds isolated from natural sources. These authors have emphasized that MIC values higher than 1 mg mL− 1 for extracts, or 0.1 mg mL− 1 for pure metabolites, show that the tested extract or metabolite is poorly active. On the other hand, activities of extract and isolated compound at concentrations below 100 μg mL− 1 and 10 μg mL− 1, respectively, are considered interesting and promising in the search of new anti-infection agents. Based on these criteria and analyzing our results (Table 1), the natural diterpenes 2, 4 and 6, as well as the semi-synthetic compounds 8 and 9 displayed MIC values lower than 10 μg mL− 1 for several evaluated Grampositive microorganisms. These results, in addition to the studies previously reported in the literature about the antimicrobial activity of pimarane diterpenes, reinforce the great importance of this class of natural products in the search of new effective antimicrobial agents. Comparing MIC values displayed by compounds 2, 4, 8 and 9 with the value obtained for 1, and taking into account all the structural aspects of these substances, it can be inferred that the presence of a hydrogen-bond donor group (HBD) at carbon C-19 or C-3 of the ent-pimarane diterpenes skeletons is very important for their increased antimicrobial activity. Also, it is noteworthy that the presence of two HBDs at
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Table 1 In vitro antibacterial activity (MIC) of the pimarane-type diterpenes against Gram-positive bacteria. Microorganism
Bacillus cereus Bacillus subtilis Citrobacter freundii Enterobacter aerogenes Enterococcus faecalis Enterococcus hirae Escherichia coli Kocuria rhizophila Morganella morganii Proteus mirabilis Pseudomonas aeruginosa Shigella flexneri Staphylococcus aureus a Staphylococcus aureus b Staphylococcus aureus c Staphylococcus epidermidis Streptococcus agalactiae Streptococcus dysgalactiae Streptococcus pneumoniae Streptococcus pyogenes
Minimum inhibitory concentration [μg mL− 1 (μM)] Van
Strep
VaDRE 1
7
8
9
0.1 (0.07) 0.1 (0.07) – 0.7 (0.47)
– – 0.7 (0.48) –
20.0 40.0 ⁎ ⁎
5.0 (16.53) 5.0 (16.53) n.e. n.e. n.e. n.e.
⁎ ⁎
10.0 (34.67) 10.0 (34.67) n.e. n.e. n.e. n.e.
⁎ ⁎
10.0 (32.63) 12.0 (39.15) n.e. n.e. n.e. n.e.
50.0 (157.01) 60.0 (188.41) n.e. n.e.
10.0 (30.26) 12.0 (36.31) n.e. n.e.
3.5 (10.79) 10.0 (30.82) n.e. n.e.
0.3 (0.20) 0.4 (0.27) – 0.1 (0.07) – – –
– – 5.9 (4.05) – 5.9 (4.05) 5.9 (4.05) 5.9 (4.05)
60.0 80.0 ⁎
⁎ ⁎
⁎ ⁎
⁎ ⁎
⁎ ⁎
⁎
10.0 (30.82) 15.0 (46.23) n.e. 10.0 (30.82) n.e. n.e. n.e.
50.0 ⁎ ⁎ ⁎
2
⁎ ⁎
n.e. ⁎ n.e. n.e. n.e.
15.0 (49.59) 15.0 (49.59) n.e. 10.0 (33.06) n.e. n.e. n.e.
3
n.e. ⁎ n.e. n.e. n.e.
4
5
20.0 (69.33) 10.0 (34.67) n.e. 10.0 (34.67) n.e. n.e. n.e.
n.e. ⁎ n.e. n.e. n.e.
6
⁎ 20.0 (65.26) n.e. 15.0 (48.94) n.e. n.e. n.e.
n.e. 30.0 (94.21) n.e. n.e. n.e.
15.0 (45.39) n.e. 12.0 (36.31) n.e. n.e. n.e.
n.e. ⁎
n.e. n.e. 80.0 (242.05) 10.0 (30.82)
– 1.5 (1.03) 0.4 (0.27) –
⁎ 90.0
n.e. n.e. ⁎ 20.0 (66.13)
0.5 (0.34) –
80.0
⁎
10.0 (33.06) ⁎
20.0 (69.33) ⁎
90.0 (293.65) ⁎
60.0 (181.54)
5.0 (15.41)
0.5 (0.34) –
80.0
⁎
10.0 (33.06) ⁎
20.0 (69.33) ⁎
90.0 (293.65) ⁎
60.0 (181.54)
10.0 (30.82)
0.5 (0.34) –
15.0
⁎
1.5 (4.96)
⁎
1.5 (5.20)
⁎
2.0 (6.53)
8.0 (25.12)
8.0 (24.21)
0.5 (1.54)
–
15.0
⁎
1.0 (3.31)
⁎
1.5 (5.20)
⁎
2.5 (8.16)
16.0 (50.24)
4.0 (12.10)
1.5 (4.62)
0.4 (0.27) –
20.0
⁎
1.0 (3.31)
⁎
1.0 (3.47)
⁎
5.0 (16.31)
12.0 (37.68)
6.0 (18.15)
0.5 (1.54)
0.2 (0.13)
–
50.0
⁎
2.0 (6.61)
⁎
3.5 (12.13)
⁎
8.0 (26.10)
15.0 (47.10)
5.0 (15.13)
1.0 (3.08)
0.1 (0.07)
–
50.0
⁎
3.0 (9.92)
⁎
4.5 (15.60)
⁎
10.0 (32.63)
20.0 (62.80)
8.0 (24.21)
1.5 (4.62)
0.7 (0.47)
n.e. n.e. n.e. n.e. ⁎ 20.0 (69.33) ⁎ 60.0 (195.77)
n.e. — not evaluated; ⁎ — Inactive in the evaluated concentrations; Vancomycin hydrochloride (Van) - positive control against Gram-positive bacteria; Streptomicyn sulfate (Strep) positive control against Gram-negative bacteria; Negative control (4% DMSO solution) did not affect the growth of the microorganisms. a ATCC — 9144. b ATCC — 6538. c ATCC — 29213.
carbons C-19/C-3 (5) and at carbons C-19/C-7 (3 and 7) causes a large decrease in the activity. Moreover, considering the intermediate MIC value displayed by 6, which contains two HBDs at carbons C-19 and C-8, it is possible to suggest that the distance between these hydrophilic groups considerably influences the antimicrobial activity displayed by this class of compounds. The same relationships were also observed by our group during the investigation of the antibacterial activities of ent-pimarane diterpenes against oral pathogens [26], suggesting that this can be a general rule for the activity of ent-pimarane diterpenes against Gram-positive microorganisms. Our results are in complete agreement with those previously reported by Urzúa et al. [40], who established that a lipophilic decalin ring system with one strategically positioned HBD is very important for the antimicrobial activity displayed by diterpenes. In this same study, the authors pointed out that a second HBD introduced in the decalin ring system leads to a reduction in or suppression of the activity. However, published data [37,41] report on significant MIC values (3.1 μg mL− 1 against M. tuberculosis and 87 μM against S. aureus) for the pimarane diterpene diaporthein B, which contains more than one HBD insertion in the decalin ring system. This fact led us to conclude that the antimicrobial
activity displayed by pimarane-type diterpenes is also ruled by structural factors other than the HBD issue. This highlights the need for further investigations on the antimicrobial activity of other pimaranes, so that their structure–activity relationship can be established. This is the first time that such a number of pimarane diterpenes were tested against a panel of microorganisms associated with several pathologies. The results obtained in this work suggest that pimaranes are an important class of natural products for further investigation in the search of new antibacterial agents. Compounds 2, 4, 6, 8, and 9 displayed very promising MIC values for most Gram-positive bacteria. Other studies, such as mammalian cell cytotoxicity, in vivo experiments, synergistic studies and evaluation against resistant bacteria are being performed by our research group to well establish their efficacy. Moreover, other classes of diterpenes are also being evaluated aiming to better understand the structure–activity relationship displayed by these compounds against Gram-positive bacteria. Acknowledgements The authors thank the FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for funds and grants (processes no. 2007/54762-8 and 2007/59017-9).
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