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Antibacterial diterpenoids from Cedrus atlantica M. Dakir
a b a
b
b
b
, F. El Hanbali , F. Mellouki , M. Akssira , A. c
Benharref , J. F. QuIlez del Moral & A. F. Barrero
c
a
Laboratoire de Chimie des Substances Naturelles , Département de Chimie Faculté des Sciences-Semlalia , Marrakech, Maroc b
Laboratoire de Chimie Bio-organique et Analytique , Faculté des Sciences et Techniques , Mohammedia, Maroc c
Departamento de Química Orgánica , Instituto de Biotecnología, facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n , 18071-Granada, Spain Published online: 20 Aug 2006.
To cite this article: M. Dakir , F. El Hanbali , F. Mellouki , M. Akssira , A. Benharref , J. F. QuIlez del Moral & A. F. Barrero (2005) Antibacterial diterpenoids from Cedrus atlantica , Natural Product Research: Formerly Natural Product Letters, 19:7, 719-722, DOI: 10.1080/14786410512331330675 To link to this article: http://dx.doi.org/10.1080/14786410512331330675
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Natural Product Research, Vol. 19, No. 7, October 2005, 719–722
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Antibacterial diterpenoids from Cedrus atlantica M. DAKIRyz, F. EL HANBALIz, F. MELLOUKIz, M. AKSSIRAz*, A. BENHARREFy, J. F. QUILEZ DEL MORALx and A. F. BARREROx yLaboratoire de Chimie des Substances Naturelles, De´partement de Chimie Faculte´ des Sciences-Semlalia, Marrakech, Maroc zLaboratoire de Chimie Bio-organique et Analytique, Faculte´ des Sciences et Techniques, Mohammedia, Maroc xDepartamento de Quı´ mica Orga´nica, Instituto de Biotecnologı´ a, facultad de Ciencias, Universidad de Granada, Campus Fuentenueva s/n, 18071-Granada, Spain (Received 1 February 2004; in final form 28 June 2004) Four diterpene alcohols were isolated from the neutral hexane extract of the cones of Cedrus atlantica, and their structures were confirmed after comparing their spectral data with literature values. These products exhibited significant antibacterial activity against gram () bacteria.
1. Introduction Diterpenoids represent a large group of natural compounds with highly interesting biological activities as antiviral, antimicrobial and anti-inflammatory agents [1–4]. Within the framework of our research program on Cedrus atlantica (Endl.), we are interested not only in analyzing the chemical composition of this species, but also in studying the biological activity and reactivity of its components. In this context, investigations were conducted on the antibacterial activity of four abietane diterpenoids, isolated from this plant. Cedrus atlantica (Endl.) is an endemic species of Morocco and Algeria [5]. This tree has a high quality wood, highly estimated by saw loggers. Its essential oil is of economical importance as a flavouring agent in perfumery and cosmetology. Previous reports on the chemical composition of this plant had led to the isolation from the acid fractions of the cones of seven acids with diterpenic skeleton, namely sandaracopimaric, abietic, isopimaric, levopimaric, palustric, dehydroabietic, and neoabietic acid [6]. Herein, as a result of our investigations on the neutral fraction of the n-hexane extract of the cones of Cedrus atlantica, we report the antibacterial activity of four alcohol diterpenoids found in this extract, 18-norabieta-8,11,13-trien-4-ol (1) [7], 7-oxodehydroabietinol (2) [8], 8,11,13-abietatriene-15,18-diol (3) [9,10] and 7 -hydroxydehydroabietinol (4) [11]. The last three compounds are dehydroabietinol derivatives, and all the compounds
*Corresponding author. Email:
[email protected] Natural Product Research ISSN 1478-6419 print: ISSN 1029-2349 online ß 2005 Taylor & Francis Group Ltd http://www.tandf.co.uk/journals DOI: 10.1080/14786410512331330675
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possess a phenyl moiety in common. Their structures were confirmed after comparing their spectral data with those found in the literature [7–10].
O
OH
CH2OH
1
2 OH
O CH2OH
3
OH CH2OH
4
1.1. Plant material The cones used in this study were collected from the region of Ifrane in the Middle Atlas Mountain during the month of August, 2002. The botanical identification was achieved by the National Scientific Institute (Rabat) where voucher specimens were deposited in the herbarium. 1.2. Extraction The plant material (715 g) was powdered and extracted in a Soxhlet apparatus with n-hexane and the resulting extract (50 g, 7%) was defatted with MeOH and fractionated with 1 N NaOH. The neutral part (12 g) was subjected to column chromatography on silica gel, using hexane/tert-buthyl methyl ether (TBME) mixtures of increasing polarity to give nine fractions (F1–F9). Fraction F5 (500 mg) was further chromatographied using hexane/TBME (8/2) to yield compound 1 (100 mg). Fraction F6 (900 mg), was rechromatographied using hexane/TBME (7/3) to give compound 2 (190 mg). Successive column chromatographies on silica gel of fraction F8 (1.8 g), with hexane/ TBME (6/4), led to the isolation of 120 mg of compound 3 and 360 mg of 4. 1.3. Antibacterial activity The qualitative antibacterial assay of the products 1–4 was carried out by the disc diffusion method [12], performed using an 18 h culture growth at 37 C and adjusted to approximately 106 CFU/mL. 500 mL of the inoculums were spread over plates containing Mu¨ller–Hinton Agar and a paper filter disc (6 mm) impregnated with 50 or 100 mg/disc of the product was placed on the surface of the media. The plates were left for 30 min at room temperature to allow the diffusion of the products, and
Antibacterial diterpenoids from C. atlantica
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Table 1. Antibacterial activities of compounds 1–4 and standard antibiotic Penicillin G. 1 Microorganisms
a
2
3
4
P
50
100
50
100
50
100
50
100
10
7 8 7 8 6 6
10 12 8 12 7 6
8 9 7 9 7 6
10 12 8 13 8 6
8 8 7 6 7 6
11 10 8 6 8 6
8 8 6 6 7 6
10 11 7 6 8 6
13 19 13 16 9 9
Bacillus cereus Streptococcus C Enterococcus faecalis Escherichia coli Proteus vulgaris Pseudomonas aeroginosa
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a ¼ mg/disc; P ¼ Penicillin G (10 unit).
then they were incubated at 37 C for 24 h. After this time, the inhibition zone around the disc was measured with a pair of calipers. Two controls were also included in the test. The first was a control involving the presence of dimethyl sulfoxide and the second, a standard antibiotic (Penicillin G) used in order to control the sensitivity of the tested microorganism. Each test was carried out in triplicate. The microorganisms tested in this study were provided from the culture collections of the Pasteur Institute (Casablanca). In the present study, Bacillus cereus (IPL 58605), Streptococcus C (IPT 2-035), Enterococcus faecalis (CIP 103214), Escherichia coli (CIP 54127), Proteus vulgaris (CIP 58605) and Pseudomonas aeroginosa (CIP A 22) were used. 2. Results and discussion Table 1 shows in vitro bacteriostatic activity of four diterpenoids of C. atlantica and the inhibition zones formed by the standard antibiotic disc. These compounds manifested significant activity against Bacillus cereus, Streptococcus C and Enterococcus faecalis, with the other bacteria being inhibited to a lesser extent, and only Pseudomonas aeroginosa remaining unaffected. This bacteriostatic property is suspected to be associated with the similar chemical profiles of these compounds. Inhibition of gram þ bacteria, E. coli and P. vulgaris bacteria may be due to the presence of the phenyl group and/or the alcohol function. In order to determine the functions responsible for this activity, the acetylated derivatives of the reported compounds will be tested in our future work. Acknowledgements This work was supported by PROTARS I (P2T2/07) and Junta Andalusia, (Consejerı´ a de Presidencia, Programa de Cooperacio´n Andalucı´ a-Marruecos). We warmly thank Dr Ali Haı¨ dour for his help and Dr A. Ouyahya for the identification of plant material. References [1] H. Ohtsu, R. Tanaka, l. yasuko, S. Matsunaga, H. Tokuda, H. Nishino, Planta Med., 67, 55 (2001). [2] A.F. Barrero, J.Q. Del Moral, R. Lucas, M. Paya´, M. Akssira, S. Akkad, F. Mellouki, J. Nat. Prod., 66, 844 (2003).
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[3] Y. Kinouchi, H. Ohtsu, H. Tokuda, H. Nishino, S. Matsunaga, R. Tanaka, J. Nat. Prod., 63, 817 (2000). [4] B. De Las Heras, J. Hoult, Planta Med., 60(6), 501 (1994). [5] (a) M. Fennane, M. Ibn Tatou, J. Mathez, A. Ouyahya, J. El Oualidi, Flore pratique du Maroc., Okad, (Ed.), Vol. 1, pp. 58–60 (1999). (b) E. Jahandiez, R. Maire, Catalogue des Plantes du Maroc., Vol. 1, p.13, Imprimerie Minerva, Alger (1931). [6] T. Norin, B. Winell, Acta. Chem. Scand., 17, 1828 (1963). [7] C.-K. Lee, J.-M. Fang, Y.-S. Cheng, Phytochemistry, 39(2), 391 (1995). [8] R. Tanaka, H. Ohtsu, S. Matsunaga, Phytochemistry, 46(6), 1051 (1997). [9] A.H. Conner, J.W. Rowe, Phytochemistry, 16, 1777 (1977). [10] A.R. Jurgens, J.D. McChesney, Magn. Reson. Chem., 28, 181 (1990). [11] A.F. Barrero, J.F. Sanchez, E.J. Alvarez-Manzaneda, M. Mun˜oz, A. Haı¨ dor, Phytochemistry, 31(2), 615 (1992). [12] P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolke, Manual of Clinical Microbiology, Vol. 6, ASM, Washington DC (1995).
